Methods of Producing Antibodies Specific for p95

ABSTRACT

The invention provides methods of measuring and/or quantifying the presence and/or amount of p95 and/or p95 complex in a sample. The invention also provides antibodies specific for p95, and methods of making such antibodies.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.13/911,329, filed Jun. 6, 2013, which is a divisional of U.S. patentapplication Ser. No. 12/629,037, filed Dec. 1, 2009, issued as U.S. Pat.No. 8,470,542 on Jun. 25, 2013, which claims the benefit of U.S.Provisional Application No. 61/118,975, filed Dec. 1, 2008, U.S.Provisional Application No. 61/182,282, filed May 29, 2009, and U.S.Provisional Application No. 61/187,960, filed Jun. 17, 2009, thedisclosures of each of which are incorporated by reference herein intheir entireties.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file SequenceListing 57618-946380.txt,created on Jun. 11, 2015, 3,125 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND OF THE INVENTION

A biomarker is generally a characteristic that is objectively measuredand evaluated as an indicator of normal biological processes, pathogenicprocesses or pharmacological responses to a therapeutic intervention.See Atkinson et al., 2001, Clin. Pharmacol. Ther. 69:89-95. Biomarkersvary widely in nature, ease of measurement and correlation withphysiological states of interest. See, e.g., Frank et al., 2003, NatureReviews Drug Discovery 2:566-580. It is widely believed that thedevelopment of new validated biomarkers will lead both to significantreductions in healthcare and drug development costs and to significantimprovements in treatment for a wide variety of diseases and conditions.Thus, a great deal of effort has been directed to using new technologiesto find new classes of biomarkers. See, e.g., Petricoin et al., 2002,Nature Reviews Drug Discovery, 1:683-695; and Sidransky, 2002, NatureReviews Cancer 2:210-219.

The interactions of cell surface membrane components play crucial rolesin transmitting extracellular signals to a cell in normal physiology andin disease conditions. In particular, many types of cell surfacereceptors undergo dimerization, oligomerization or clustering inconnection with the transduction of an extracellular event or signalinto a cellular response, such as, e.g., proliferation, increased ordecreased gene expression or the like. See, e.g., George et al., 2002,Nature Reviews Drug Discovery 1:808-820; Mellado et al, 2001, Ann. Rev.Immunol. 19:397-421; Schlessinger, 2000, Cell 103:211-225; and Yarden,2001, Eur. J. Cancer 37:S3-S8. The role of such events in diseases, suchas cancer, has been the object of intense research and has led to thedevelopment of several new drugs and drug candidates. See, e.g., Herbstand Shin, 2002, Cancer 94:1593-1611; Yarden and Sliwkowski, 2001, NatureReviews Molecular Cell Biology 2:127-137; McCormick, 1999, Trends inCell Biology 9:53-56 (1999); and Blume-Jensen and Hunter, 2001, Nature411:355-365.

Expression levels of individual cell surface receptors, such as Her-2 inbreast cancer, have been used as biomarkers, especially to determinepatient prognosis or whether a patient will or will not respond tocertain treatments. Conventional immunohistochemical (IHC) orfluorescence in situ hybridization (FISH) analyses have been used todetect Her-2 overexpression to determine whether treatment with aHer2-acting agent, e.g., trastuzumab, is warranted. Unfortunately, IHCand FISH have certain limitations as diagnostic tools in that they arenot necessarily accurate and also prone to different interpretations bydifferent laboratory personnel. Her-2 is also over-expressed in othercancers such as ovarian cancer, non-small cell lung cancer, coloncancer, prostate cancer and pancreatic cancer. See Mosession et al.,2004, Semin. Cancer. Biol. 14:262-270.

A subgroup of Her-2-overexpressing tumors also have p95Her-2 (p95), anN-terminal truncated version of Her-2 that has shed the ectodomain, towhich trastuzumab binds. Data suggest that the presence of p95correlates to the extent of lymph node involvement, suggesting that p95may be an important prognostic factor for breast cancer metastases. SeeMolina et al., 2002, Clin. Can. Res. 8:347-353. Interestingly,trastuzumab binds Her-2 but cannot bind the p95 truncated Her-2 sotrastuzumab is ineffective in patients with high levels of p95.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or amount of p95 and/or p95 complexin a sample, the method comprising providing a sample and determiningthe presence and/or quantity of p95 and/or p95 complex in the sample. Ina preferred embodiment, the sample is a biological sample. In apreferred embodiment, the sample is a tissue sample. In a preferredembodiment, the sample is a fixed sample, a frozen sample or a lysate.In a preferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample contains exosomes and/or othervesicles. In a preferred embodiment, the sample comprises cell lines. Ina preferred embodiment, the measurement may be quantitative across awide dynamic range.

In a second aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or quantity of p95 and/or p95complex in a sample, the method comprising mixing a sample with abinding compound and determining the presence and/or quantity of bindingcompound bound to p95 and/or p95 complex. In a preferred embodiment, thebinding compound is capable of specifically binding p95. In a preferredembodiment, the binding compound comprises an antibody. In a preferredembodiment, the antibody was raised against one of the peptides havingSEQ ID NOs 1-7.

SEQ ID NO 1 MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIIS SEQ ID NO 2ASPLTSIIS SEQ ID NO 3 PAEQRASPLTSIIS SEQ ID NO 4 QPCPINCTHSCVDLDDKGCPASEQ ID NO 5 MPIWKFPDEEGA SEQ ID NO 6 PSGVKPDLSYMPIWK SEQ ID NO: 7Ac-QPCPINCTHSCVDLDDKGCPAKK(εNH)-[KLH] (shown as conjugated to KLH)

In certain embodiments, the antibody is or comprises one of theantibodies produced by hybridoma cell lines deposited with the ATCChaving accession number PTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) andPTA-9740 (p95.D9.1). In one embodiment, the antibody is p95.D9.1. In apreferred embodiment, the sample is a biological sample. In a preferredembodiment, the sample is a tissue sample. In a preferred embodiment,the sample is a fixed sample, a frozen sample or a lysate. In apreferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample contains exosomes and/or othervesicles. In a preferred embodiment, the sample comprises cell lines. Ina preferred embodiment, the measurement may be quantitative across awide dynamic range.

In a preferred embodiment, determining the presence and/or quantity ofbinding compound bound to p95 further comprises providing a secondbinding compound, the second binding compound being able to specificallybind the binding compound bound to p95 and determining the presenceand/or quantity of the second binding compound as correlative of thepresence and/or quantity of the binding compound bound to p95. In apreferred embodiment, the second binding compound is an antibody.

In a third aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or quantity of p95 and/or a p95complex in a sample, the method comprising: mixing (i) a sample that maycontain p95 and/or p95 complex; (ii) a proximity probe that is capableof binding p95 and/or at least one other analyte in a p95 complex, theproximity probe having an effective proximity; and (iii) at least onebinding compound, the at least one binding compound being capable ofbinding p95 and/or at least one other analyte and having one or moresignaling molecules attached, wherein binding of the proximity probe andbinding compound within the effective proximity produces a signal fromthe molecular tags that correlates with the presence and/or quantity ofp95 and/or the p95 complex. In a preferred embodiment, the proximityprobe and/or binding compound is capable of specifically binding p95 orthe at least one other analyte. In a preferred embodiment, the proximityprobe and/or binding compound further comprises an antibody. In apreferred embodiment, the proximity probe and/or the binding compoundfurther comprises an antibody, and each antibody binds to a specificepitope on p95. In a preferred embodiment, the antibody was raisedagainst one of the peptides having SEQ ID NOs 1-6. In certainembodiments, the antibody is or comprises one of the antibodies producedby hybridoma cell lines deposited with the ATCC having accession numberPTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1). In oneembodiment, the antibody is p95.D9.1.

In a preferred embodiment, the proximity probe comprises an antibody anda first nucleic acid and the binding compound comprises an antibody anda second nucleic acid, wherein the first and the second nucleic acidsare complementary to each other and able to hybridize to determine theeffective proximity and produce the signal, directly or indirectly,through hybridization. Hybridization may be quantified by any methodknown to one skilled in the art such as, for example, measuringmolecular tags attached to the nucleic acid molecules or measuringhybridization with any method known to one skilled in the art. In apreferred embodiment, hybridization is measured through a nucleic acidamplification method such as, for example, the rolling circleamplification method. In a preferred embodiment, the antibody was raisedagainst one of the peptides having SEQ ID NOs 1-7. In certainembodiments, the antibody is or comprises one of the antibodies producedby hybridoma cell lines deposited with the ATCC having accession numberPTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1). In oneembodiment, the antibody is p95.D9.1 In a preferred embodiment, thesample is a biological sample. In a preferred embodiment, the sample isa tissue sample. In a preferred embodiment, the sample is a fixedsample, a frozen sample or a lysate. In a preferred embodiment, thesample is a tumor sample. In a preferred embodiment, the sample is afrozen tumor tissue sample. In a preferred embodiment, the samplecomprises a tumor lysate. In a preferred embodiment, the samplecomprises a breast cancer sample. In a preferred embodiment, the sampleis an FFPE sample. In a preferred embodiment, the sample is a blood,plasma or lymph sample. In a preferred embodiment, the blood or plasmasample contains circulating tumor cells. In a preferred embodiment, thesample contains exosomes and/or other vesicles. In a preferredembodiment, the sample comprises cell lines. In a preferred embodiment,the measurement may be quantitative across a wide dynamic range.

In a preferred embodiment, the proximity probe comprises a cleavingprobe that has a cleavage inducing moiety and the at least one bindingcompound has one or more molecular tags attached to the binding compoundby a cleavable linkage, wherein the cleavable linkage may be cleavedwithin the effective proximity producing a signal that correlates withthe presence and/or quantity of p95 and/or p95 complex. In a preferredembodiment, the binding compound and/or the cleaving probe furthercomprises an antibody, and each antibody binds to a specific epitope onp95 and/or at least one other analyte in a p95 complex. In a preferredembodiment, the antibody was raised against one of the peptides havingSEQ ID NOs 1-7. In certain embodiments, the antibody is or comprises oneof the antibodies produced by hybridoma cell lines deposited with theATCC having accession number PTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2)and PTA-9740 (p95.D9.1). In one embodiment, the antibody is p95.D9.1. Ina preferred embodiment, the sample is a biological sample. In apreferred embodiment, the sample is a tissue sample. In a preferredembodiment, the sample is a fixed sample, a frozen sample or a lysate.In a preferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample contains exosomes and/or othervesicles. In a preferred embodiment, the sample comprises cell lines. Ina preferred embodiment, the measurement is quantitative across a widedynamic range.

In a fourth aspect, the invention is drawn to a purified antibody thatbinds to p95. In a preferred embodiment, the purified antibody bindsspecifically to p95. In a preferred embodiment, the antibody bindsspecifically to the extracellular domain of p95 but not full lengthHER2. In a preferred embodiment, the antibody is a polyclonal antibodyor a monoclonal antibody. In a preferred embodiment, the antibody is amonoclonal antibody. In a preferred embodiment, the antibody was raisedagainst one of the peptides having SEQ ID NOs 1-7. In certainembodiments, the antibody is or comprises one of the antibodies producedby hybridoma cell lines deposited with the ATCC having accession numberPTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1). In oneembodiment, the antibody is p95.D9.1.

In a fifth aspect, the invention is drawn to a method for determiningwhether a subject with a cancer is likely to respond to treatment with atargeted therapy, for predicting a time course of disease and/or forpredicting probability of a significant event in the time course of thesubject's cancer based on a measurement of an amount of p95 and/or a p95complex in a sample. In one embodiment, the invention is drawn to amethod for determining whether a subject with a cancer is likely torespond to treatment with a Her-2 acting agent. In another embodiment,the method is drawn to a method of predicting a time course of a diseasein a subject with a cancer. In another embodiment, the method is drawnto predicting the probability of a significant event in a subject with acancer.

In a preferred embodiment, a time course is measured by determining thetime between significant events in the course of a patient's disease,wherein the measurement is predictive of whether a patient has a longtime course. In a preferred embodiment, the significant event is theprogression from primary diagnosis to death. In a preferred embodiment,the significant event is the progression from primary diagnosis tometastatic disease. In a preferred embodiment, the significant event isthe progression from primary diagnosis to relapse. In a preferredembodiment, the significant event is the progression from surgery todeath. In a preferred embodiment, the significant event is theprogression from surgery to metastases. In a preferred embodiment, thesignificant event is the progression from surgery to relapse. In apreferred embodiment, the significant event is the progression frommetastatic disease to death. In a preferred embodiment, the significantevent is the progression from primary diagnosis to relapse. In apreferred embodiment, the significant event is the progression frommetastatic disease to relapse. In a preferred embodiment, thesignificant event is the progression from relapse to death. In apreferred embodiment, the time course is measured with respect tooverall survival rate, time to progression and/or using the RECIST orother response criteria.

In a preferred embodiment, the subject's cancer is breast cancer. In apreferred embodiment, the targeted therapy comprises a Her-2 actingagent. In a preferred embodiment, the Her-2 acting agent is trastuzumaband/or pertuzumab. In a preferred embodiment, the Her-2 acting agent isa tyrosine kinase inhibitor and if the amount of p95 is high, then thepatient is likely to respond to the targeted therapy, the patient islikely to have a long time course and/or the patient is not likely tohave a significant event. In a preferred embodiment, the Her-2 actingagent is lapatinib. In a preferred embodiment, the targeted therapy isan inhibitor, such as a protease inhibitor, and if the amount of p95 ishigh, then the patient is likely to respond to the targeted therapy, thepatient is likely to have a long time course and/or the patient is notlikely to have a significant event. In a preferred embodiment, theinhibitor inhibits metalloproteases including, but not limited to,matrix metalloproteases and/or member(s) of the ADAM family ofproteases. In a preferred embodiment, the inhibitor inhibits ADAM10.

In a preferred embodiment, determining whether an amount of p95 is lowis done by comparing the amount of p95 in the subject's cancer to anoptimal cutoff. Such optimal cutoffs are disclosed herein, and certainembodiments of the invention are meant to include amounts that areapproximate to the amounts mentioned and disclosed herein. In certainembodiments, the amount of p95 in the subject is compared to an optimalcutoff, and the optimal cutoff is used to determine whether a patientwill respond to an appropriate treatment.

In a further aspect, the invention provides methods of treating asubject with cancer. In one aspect, the methods comprise determiningthat the subject is afflicted with a cancer that is likely to respond totreatment and/or has a long time course according to a method of theinvention, and administering an effective amount of compound to thesubject as a result of said determination. In another aspect, themethods comprise determining that a subject is afflicted with a cancerthat is likely to respond to treatment according to a method of theinvention, then advising a medical professional of the treatment optionof administering to the subject an effective amount of an agent. Inanother aspect, the agent is at least two agents and the medicalprofessional is advised of treatment options based upon the methods ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ELISA data for antibodies generated against a p95 peptide(shown as SEQ ID No. 5). Conditioned media from individual hybridomaclones (D1-D13, listed in column 1) were tested in ELISA assays againstthe p95 peptide used for immunizing the mice from which the hybridomaswere derived (labeled Her2-D pep, shown in column 2), the HER2extracellular domain (HER2-ECD, labeled as Her2-hFc, shown in column 3),and a peptide different from that used in immunization (labeled asHer2-A pep, shown in column 4). Positive controls for both the HER2-ECDand the Her2-A peptide are also shown. Highlighted cells in the tableshow positive reactivity in the ELISA test. Several clones showreactivity to the p95 peptide used for immunization but littlereactivity with the HER2-ECD (clones D3, D5, D7-11 and D13). Others showreactivity to both the p95 peptide and the HER2-ECD (clones D4, D6, andD12).

FIG. 2 shows Western blot screening of hybridoma conditioned media. 5 ugcell lysate from either 293T cells (lane 3) and SKBR3 cells (lane 2) and1 ug cell lysate from 293T cells transfected with pcDNA6-p95 (anexpression vector for p95, lane 1) were separated on 4-12% NuPAGE gels(Invitrogen). The gels were blotted to PVDF membranes that were stainedwith conditioned media from hybridomas D4, D8, D12 or Her2 Ab8. Her2 Ab8(Labvision, Fremont, Calif.) was used as positive control antibody andbinds to an intracellular epitope of Her2 that is also part ofpcDNA6-p95. Bound antibodies were detected with a horseradishperoxidase-conjugated anti-mouse IgG antiserum and an ECL reagent.

FIGS. 3 a and 3 b show fluorescence-activated cell sorting (FACS)results from both native cells (FIG. 3 a) and cells that werepermeabilized and fixed (FIG. 3 b). Each panel shows the results oftransfected 293T cells bound to conditioned media from different clones(D4, D8, D9 and D12 in the top rows of each figure; D3, D7, D10 and D11in the bottom rows). Bound antibody was detected with a biotinylatedanti-mouse antibody followed by streptavidin-PE. The two panels on theleft side show 293T cells transfected with pcDNA6-HER2, which expressesthe full length HER2 protein; the two panels in the middle show 293cells transfected with pcDNA6myc/hisA M611-p95, which expresses p95.Both HER2 and p95 expression proteins have an N-terminal hemagglutinintag. The two panels on the right side show 293T cells transfected with avector that expresses an irrelevant protein. Each panel also shows apositive control (HA.A28.2, an antibody directed to the N-terminalhemagglutinin tags on the expressed p95 and HER2 proteins) and anegative control (an anti-ricin antibody). The y-axis is a histogramshowing the number of events at a particular bin of PE signal on thex-axis. The binding characteristics for native vs. fixed cells weresimilar (except for D11), suggesting that formalin-fixedparaffin-embedded (FFPE) samples may show similar bindingcharacteristics. Antibodies that bound strongly to p95 and weakly toHER2 included clones D4, D8, D9 and D12. Antibodies that bound stronglyto p95 but not to HER2 included clones D3, D7, D10 and D11.

FIG. 4 shows Western blot data for the cells used subsequently increating formalin-fixed paraffin-embedded (FFPE) blocks. Cell lysateswere prepared from samples removed just prior to the addition offixative. Proteins in these cell lysates were separated using PAGE,transferred to nitrocellulose membranes, blocked, probed with anti-HER2Ab8 and detected using a horseradish peroxidase detection kit. Lanes 1-4show cell lysates from MCF-7-HER2c, MCF-7-p95c, SKBR3-p95c andSKBR3-p95c2, respectively. These cells were obtained from the laboratoryof Jose Baselga; they express proteins that contain no leader sequenceor HA-tag. Lanes 5-8 show lysates from MCF-7 cells (lane 5) and MCF-7cells transiently transfected with a 50:50 weight mix of pcDNA6-HER2 andempty vector (lane 6), a 50:50 weight mix of pcDNA6-HER2 andpcDNA6myc/hisA M611-p95 (lane 7) or 100% pcDNA6myc/hisA M611-p95 (lane8). Lane 9 shows cell lysate from SKBR3 cells.

FIG. 5 a shows the work flow and assay configuration of a p95 VeraTagassay on FFPE samples. The upper left section of the figure outlines theworkflow for the VeraTag assay. A slide containing the sample ofinterest is de-paraffinized and rehydrated, then blocked and p95antibody is added. A second antibody labeled with VeraTag (i.e.,“anti-mouse secondary”) is added that binds to the first antibody, asshown in the right part of FIG. 5 a. The sample is rinsed and VeraTag isreleased, captured and measured using capillary electrophoresis (CE).The lower panel of FIG. 5 a shows a typical CE electropherogram.

Proprietary software (VeraTag Informer Software) is used to evaluate theraw electropherograms. The software integrates fluorescent peaksassociated with the released tag as well as an internal standard,fluorescein of known concentration, used for capillary normalization togive a relative peak area (VeraTag peak area/fluorescein peak area).Separately, the tumor area revealed by hematoxylin and eosin staining isassessed by a pathologist. The relative peak area is divided by thistumor area to give the final value.

The reproducibility of the assay outlined in FIG. 5 a is shown in FIG. 5b using 3 types of comparisons. The upper left panel shows a comparisonof assay data accrued for 19 tumor samples on one day (x-axis, 8/19 p95levels) vs. a second set of data on the same samples accrued on anotherday (y-axis, 8/22 p95 levels). The upper right panel show analyses of aset of 12 tumor samples performed on 3 different days (y-axis, p95levels) compared to the mean p95 level (x-axis). The bottom panel ofFIG. 5 b shows the combined data for all 5 experimental sets (y-axis,p95 levels) compared with the mean p95 level (x-axis, p95 levels). Ineach panel, the diagonal line represents perfect correlation between x-and y-axis data, which is expressed as relative peak area multiplied byuL per cm² (RPA*uL/sqcm).

Consistency between operators and over time is critical for standardizedassays. FIG. 5 c shows how batch consistency was achieved using celllines with widely varying amounts of p95 as normalization standards. Ineach of the first three top panels, labeled “PRE-normalization”,pair-wise comparisons between 3 operators are shown. In each case, theunits of p95 are shown as relative peak areas multiplied by uL per cm²(RPA*uL/sqcm). On the top right panel, the same scores of two operatorsare plotted against the scores acquired by a third operator afternormalization to internal standards. The process for normalization isdescribed in Example 4. The bottom panel shows the coefficient ofvariability (CV, y-axis) between both batches/operators over a widerange of p95 levels (mean p95 plotted on the x-axis).

FIG. 6 shows the results of a VeraTag assay using different purifiedantibodies against MCF-7 and SKBR3 cells transfected with a C-terminalfragment (CTF) of HER2, p95 or full length HER2. HER2 CTFs, found inboth the cytoplasm and nucleus, are generated by alternative initiationof translation from methionines located near the transmembrane domain ofthe full-length molecule. Like HER2 and p95, tumors dependent on CTFsare sensitive to kinase inhibitors and like tumors dependent on p95,they do not respond to therapeutic antibodies against HER2. Theantibodies used in the VeraTag assay were generated using a peptide (SEQID No. 5) from p95. Each purified antibody (D4.1, D7.2, D8.2, D9.1,D10.1 and D12.1 as shown on the x-axis) was tested with 6 cell lines(listed from top to bottom in the legend and displayed from left toright in each bar graph): MCF-7, MCF-7 expressing CTF, MCF-7 expressingHER2, MCF-7 expressing p95, SKBR3 and SKBR3 expressing CTF. The y-axisis shown in Relative Peak Area multiplied by μl/cm² (RPA*uL/sqcm), asset forth in Example 4. Antibody A3.1 was generated by challenging micewith an irrelevant peptide. The positive control was Ab8, which targetsthe cytoplasmic domain of HER2. Both D4.1 and D12.1 act much like thecontrol antibody. D8.2 and D9.1 show specificity for p95.

FIGS. 7 a-7 c show quantitation of p95 in 12 different tumor samples(Cohort A). The 12 tumor samples were chosen to be highly HER2 positiveand from patients with node-positive status. Half of each tumor samplewas fresh-frozen and half was formalin-fixed and paraffin-embedded. Thefresh-frozen samples were used to generate cell lysates that were testedin Western blots for the ability to bind an antibody, CB11, which wasraised against the intracellular domain of HER2. The Western blotresults are shown in FIG. 7 a. The 12 tumor samples are labeled 1 to 12,left to right. There is a marker lane between samples 7 and 8. SKBR3cell lysate was used as a positive control (right lane). The presence ofsignificant amounts of p95 is seen in samples 1, 2, 3, 5, 7, 8 and 10;these tumors were designated p95-positive.

FFPE slides from all 12 tumors, along with 7 cell standards, were testedin the VeraTag assay outlined in FIG. 5, using clone D9.1, which hasbeen shown to be p95-specific (see FIG. 6). The assay results from FFPEsamples of clones designated by Western blot using fresh-frozen samplesto be p95-positive or p95-negative are shown in FIG. 7 b. The x-axisshows p95 positive or negative by Western blot; the y-axis showsRelative Peak Area multiplied by μl/cm², as set forth in Example 4.These results have been replotted to show the results for individualclones in FIG. 7 c, alongside the results for the 7 cell standards. TheWestern-positive clones 1, 2, 3, 5, 7, 8 and 10 are shown on the left,followed by the Western-negative clones 4, 6, 9, 11 and 12, followed bythe cell standards, which are as follows: MCF-7, MCF-7-p95, MCF-7-CTF,MCF-7-HER2, SKBR3, SKBR3-CTF and MDA-MB-453. The y-axis shows p95 levelsin units of Relative Peak Area multiplied by μl/cm² (RPA*uL/sqcm), asset forth in Example 4. Especially considering the likely heterogeneitybetween the fresh frozen cells used for the Western blot and the FFPEsamples used for the assay, there is a clear correlation between the twomethods of quantifying p95.

FIGS. 8 a-8 c demonstrate the specificity of the D9.1 antibody ascompared to its isotype control. Isotype control antibodies are used toshow the non-specific binding of target primary antibodies to cellsurface antigens. The isotype control antibody used in this experimentis IgG2a, matching the isotype of D9.1. The assay described in FIG. 5was used to test both Western-positive and Western-negative FFPE tumorsamples, along with 6 cell standards. In FIG. 8 a, the Western-positiveclones 1, 2, 3, 5, 7, 8 and 10 are shown on the left, followed by theWestern-negative clones 4, 6, 9, 11 and 12, followed by the cellstandards, which are as follows: MCF-7, MCF-7-p95, MCF-7-CTF,MCF-7-HER2, SKBR3 and SKBR3-CTF. In each sample, the binding tomonoclonal antibody D9.1 is shown on the left, the binding to theisotype control antibody, IgG2a, is shown on the right. The y-axis showsp95 levels in Relative Peak Area multiplied by μl/cm² (RPA*uL/sqcm), asset forth in Example 4. In FIG. 8 b (lower left panel), the results forWestern-negative and Western-positive samples prior to subtractingnon-specific binding are shown; in FIG. 8 c (lower right panel), theresults for Western-negative and Western-positive samples are shownafter the non-specific isotype binding has been subtracted. The x-axisshows p95 negative or positive by Western blot; the y-axis shows p95levels in Relative Peak Area multiplied by μl/cm² (RPA*uL/sqcm), as setforth in Example 4. The difference in the means between p95-positive andp95-negative clones is about 4-fold with a dynamic range ofapproximately 10-fold.

FIGS. 9 a-9 d show data demonstrating that p95-positive tumors are morelikely to be highly HER2-positive, as shown by the VeraTag HER2-total(H2T) assay. The tumor samples described in FIGS. 7 and 8 were tested inthe H2T assay (Cohort A). The data are shown in FIG. 9 a. The tumorsspanned the range of the HER2 positivity, but the mean score of samplesdeemed p95-positive by Western blot data were significantly higher thanthe p95-negatives (see, the Mann-Whitney test as set forth in Conover,W. J. (1980), Practical Nonparametric statistics (3^(th) Ed.)).

A second set of 18 FFPE tumor samples (Cohort B, diamonds) was testedusing both the p95 assay as well as the H2T assay. FIG. 9 b shows thecorrelative results. Cohort A (squares) corresponds to the tumor setmeasured in FIG. 7 c. The approximate cutoff for Western positivityinferred from FIG. 7 b is shown by the arrow on the y-axis in FIG. 9 b.In general, a higher p95 signal is more likely to be found associatedwith a high H2T signal in both cohorts.

A large cohort of trastuzumab-treated patients whose tumors hadpreviously been assessed for H2T were investigated to determine if therewas any correlation between poor outcomes and samples in the high HER2range theoretically enriched for p95. This cohort was derived from theInternational Serum Her2/neu Study Group (ISHSG) and is called theLipton cohort. These patients were selected primarily by IHC performedat a central location—the University of Vienna in Austria—by a singlepathologist. 90% of the patients were IHC3+, and 80/92 receivedtrastuzumab in combination with chemotherapy while 12 receivedtrastuzumab as a single drug. 88/92 patient had metastatic breast cancerand they could have received trastuzumab either as a first, second orthird line therapy. For the high HER2 range, a cut-off value oflog₁₀(H2T)≧1.95 was established just above the highest p95-negativesample (the cut-off is shown in FIG. 9 a). Above this H2T cutoff, tumorscould be described as p95-enriched while those below the cutoff would bep95-equivocal. Among those patients confirmed to be HER2-FISH-positive,those in the p95-enriched group had significantly shortertime-to-progression (shown in FIG. 9 c) and overall survival (shown inFIG. 9 d) than those that were in the p95-equivocal group.

FIGS. 10 a and 10 b show colorimetric immunohistochemistry (IHC) of FFPEcell lines and tumor samples that are positive or negative for p95. InFIG. 10 a, both assay data (top panel) for p95 levels (expressed inRPA*uL/sqcm) and IHC data (bottom panel) of FFPE cell lines probed withD9.1 are shown. The cell lines shown are MCF7, MCF7 transfected witheither p95 or full-length Her2, SKBR3 and SKBR3 transfected with CTF,the C-terminal fragment of HER2 that is found in both the nucleus andcytoplasm. MCF7 is known to express little Her2, while the SKBR3parental cell line is known to express high amounts of full-length HER2and low levels of p95. MCF7 and SKBR3 cells stained with D9.1 showlittle staining, consistent with the low level of p95. MCF7-p95 cellsstained with D9.1 show staining localized primarily to the cellmembrane, consistent with the location of the p95. The IgG2a controlisotype antibody showed no cell membrane staining, demonstrating thespecificity of the D9 antibody (data not shown). The results of the IHCare consistent with the results seen using the VeraTag assay.

FIG. 10 b shows both assay and IHC data for FFPE tumor samples. In theleft panel, p95 assay data is shown for three tumor samples (#s 5, 6 and12), as well as two cell lines (MCF7 and MCF7 transfected with p95). Theunits for the p95 levels are expressed as RPA*uL/sqcm). In the rightpanel, IHC data is shown for FFPE samples probed with either D9.1, ap95-specific antibody (top panels) or CB11, an antibody targeted to theintracellular domain of HER2 (bottom panels). All three tumors showstaining with the CB11 antibody, consistent with the presence offull-length HER2, while only tumor 5, shown in both Western blots andthe VeraTag assay to be p95-positive, stains with D9.1. These datafurther suggest that the epitope recognized by the D9.1 antibody isfound in naturally-occurring forms of p95 found in breast tumor tissue.

FIG. 11 shows the results of quantitation studies of p95 using anti-p95antibodies labeled directly with molecular tags. The experiment wasperformed as outlined in FIG. 5, except that the antibodies were labeleddirectly with molecular tags rather than using a secondary anti-mouseantibody. The purified monoclonal antibodies used in these studies wereD4.1, D8.2, D9.1 and D12.1 as shown on the x-axis; the y-axis is shownin Relative Peak Area multiplied by μl/cm², as set forth in Example 4.The anti-HER2 antibody Ab8 was used as a positive control. The celllines tested were, from top to bottom in the legend and from left toright in the bar graph, MCF-7, MCF-7-CTF, MCF-7-her2, MCF-7-p95, SKBR3and SKBR3-CTF. The results shown are very similar to those shown in FIG.6, except for a reduction in the dynamic range between thep95-high-expressing cell lines (MCF-7-CTF, MCF-7-p95 and SKBR3-CTF) andthe p95-low-expressing cell lines (MCF-7, MCF-7-Her2 and SKBR3).

FIGS. 12 a-12 d show two sets of results using the D9.1 antibody tomeasure p95 levels in cell line standards and tumors, left and right,respectively. In FIGS. 12 a and 12 b, D9.1 is labeled with VeraTag toquantitate p95 in cell line standards (FIG. 12 a) and tumor samples fromCohort A (FIG. 12 b) using a single antibody assay. The cell linestandards tested are, from left to right on the x-axis as shown in FIG.12 a, MCF-7, MCF-7-p95, MCF-7-CTF, MCF-7-HER2, SKBR3, SKBR3-CTF andMDA-MB-453. The y-axis shows p95 levels in Relative Peak Area multipliedby μl/cm², as set forth in Example 4. In FIG. 12 b, the results of thesingle antibody assay are shown for tumor samples from Cohort A, whichhave been classified as p95-positive or p95-negative based on Westernblot data. The x-axis shows p95 negative or p95 positive cell lines; they-axis shows p95 levels in Relative Peak Area multiplied by μl/cm².

FIGS. 12 c and 12 d show the same cell line standards and tumor samplestested using a two antibody assay system in which both D9.1, which isp95-specific, and Ab8, which is specific for the intracellular domain ofHER2, are used to detect p95. The Ab8 antibody is labeled with biotin;the D9.1 antibody has a cleavable VeraTag, allowing for a proximityassay in which the presence of the two antibodies within the distance ofthe same p95 molecule causes the release of the fluorophor on theVeraTag. As the data show, separation of p95-negative and p95-positivesubgroups are retained with this form of the assay. The x-axis shows p95negative or p95 positive cell lines; the y-axis shows p95 levels inRelative Peak Area multiplied by μl/cm².

FIG. 13 shows the growth inhibition of breast cancer cell line SKBR3 andBT474 using anti-p95 antibodies compared to 4D5, the mouse version oftrastuzumab. Several monoclonal antibodies generated from micechallenged with the D peptide from p95 were tested for their ability toinhibit growth in SKBR3 and BT474 cells, both of which are known toexpress high levels of HER2. 4D5 was used as a positive control. Anantibody (A3) to an unrelated peptide was used as a negative control.The top 3 panels show results for SKBR3 cells; the bottom 3 panels showresults for BT474 cells. The x-axis shows antibody concentration; they-axis shows the difference in absorbance at 492 nM and 690 nM. In thisexperiment, cells were grown for 3 days in the presence of an antibody,then growth was assessed using the XTT assay. The results suggest thatD3.4 and D4.1 inhibit the growth of SKBR3 cells but not BT474 cells.

FIG. 14 shows a sub-population treatment effect pattern plot (STEPP),generated to examine the progression-free survival (PFS) rate at 12months after treatment with trastuzumab across the distribution of H2T.Bins of 30 patients were ordered smallest to largest H2T. A trend ofincreasing probability of remaining progression-free past 12 months wasobserved for increasing H2T. However, at the highest levels of H2T, anabrupt decrease in the PFS rate was observed, consistent with areduction in susceptibility to trastuzumab.

FIG. 15 shows a Kaplan-Meier (KM) analyses comparing the PFS of FISH(−),H2T low (log₁₀H2T<1.25) patients with those of FISH(+), H2T high(Log₁₀H2T≧1.95 and FISH(+), H2T intermediate (1.25<log₁₀H2T<1.95).Cut-offs were identified by lowest p-value in a positional scanninganalysis. KM analyses demonstrated that patients who were FISH(+), H2Tintermediate had a significantly longer PFS than patients who wereFISH(−), H2T low (median PFS 12.6 vs. 4.5 months; hazard ratio(HR)=0.34; p<0.0001). Patients that were FISH(+), H2T high experienced aPFS that was no better than patients that were FISH(−), H2T low (medianPFS 4.6 vs. 4.5 months; HR=0.87; p=0.68).

FIG. 16 shows the discrimination of patient populations by H2T and p95(N=90). Previous analyses of this cohort using VeraTag measures of HER2protein expression (H2T) identified an H2T-high subgroup with longer TTPthan the H2T-low subgroup. A Cut-off for p95 was identified by lowestp-value in a positional scanning analysis

This Figure shows Kaplan-Meier (KM) analyses comparing the % progressionfree (TTP) of H2T low (log 10H2T<1.25) patients with those of H2T high(Log 10H2T≧1.25 or linear >13.8) and low p95 (log 10H2T<90) withpatients with high H2T (Log 10H2T≧1.25) and high p95 (log 10H2T>90).

KM analyses demonstrated that patients who were H2T low had asignificantly shorter median TTP (in response to trastuzumab) thanpatients with High H2T, low 95 (median TTP 4.4 vs. 11.7; hazard ratio(HR)=2.4; p=0.0003). Patients that were high H2T, high p95 alsoexperienced a significantly shorter median TTP compared with patientswith High H2T/low p95 (median TTP 7.2 vs 11.7 months; hazard ratio(HR)=1.9, p=0.017). Similar results were seen with overall survival,such that KM analyses for OS demonstrated that patients who were H2T lowhad a significantly shorter median OS (in response to trastuzumab) thanpatients with High H2T, low 95 (median OS 29 vs. 48 months; hazard ratio(HR)=1.9; p=0.042). Patients that were high H2T, high p95 alsoexperienced a significantly shorter median OS compared with patientswith High H2T/low p95 (median OS 29 months vs 48 months; hazard ratio(HR)=2.3, p=0.0095).

FIG. 17 shows Kaplan-Meier (KM) analyses comparing the percentprogression free (time to progression, TTP) of various subgroups fromthe Lipton cohort, as defined by VeraTag measurements of HER2 total (H2Thigh or low), and p95HER2 (p95 high or low). Cut-offs were identified bylowest p-value in a positional scanning analysis. H2T high=(log10H2T>1.25 or on a linear scale, >13.8). Low H2T=log 10H2T<=1.25 or on alinear scale, <=13.8. p95 low=p95<=90 and p95 high=p95>90 (on a linearscale).

KM analyses demonstrated that patients who were FISH positive, H2T high,p95 low (green line) had a (significantly) longer median TTP thanpatients who were FISH negative, H2T low (red line). Patients that wereFISH-positive, H2T low (blue line) experienced a PFS that wassuperimposable (i.e., no better) than patients that were FISH negative,H2T low (red line). In addition, patients that were FISH positive, H2Thigh, and p95 high (orange line) experienced PFS that was again, nearlysuperimposable on the other 2 less-favored groups indicated by the redand blue lines (FISH negative, H2T low and FISH positive, H2Tlow,respectively). The group with the best outcomes in this study was thegroup in green, who were FISH positive, H2T high, and p95 low.

DETAILED DESCRIPTION OF THE INVENTION

“Antibody” means an immunoglobulin that binds to, and is thereby definedas complementary with, a particular spatial and polar organization ofanother molecule. The antibody can be monoclonal, polyclonal orrecombinant and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal) or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for binding. Antibodies may include a completeimmunoglobulin or fragment thereof, which immunoglobulins include thevarious classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2band IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab′)2,Fab′ and the like. Antibodies may also be single-chain antibodies,chimeric antibodies, humanized antibodies or any other antibodyderivative known to one of skill in the art that retains bindingactivity that is specific for a particular binding site. In addition,aggregates, polymers and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular binding site is maintained. Guidance in the production andselection of antibodies and antibody derivatives for use inimmunoassays, including such assays employing releasable molecular tags(as described below) can be found in readily available texts andmanuals, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York; Howard and Bethell, 2001,Basic Methods in Antibody Production and Characterization, CRC Press;Wild, ed., 1994, The Immunoassay Handbook, Stockton Press, New York.

“Binding compound” shall refer to a molecule capable of binding toanother molecule of interest. A binding compound may be an antibody, apeptide, a peptide or non-peptide ligand for a cell surface receptor, aprotein, an oligonucleotide, an oligonucleotide analog, such as apeptide nucleic acid, a lectin or any other molecular entity that iscapable of specifically binding to a target molecule or complex. In oneembodiment, the target molecule is a protein or protein complex. Inanother embodiment, a binding compound further comprises a proximityprobe. In one embodiment, a binding compound comprises one or moremolecular tags attached to a binding moiety. In another embodiment, asecond binding compound may be bound to the binding compound andmeasured or quantified as a correlative for the presence of the bindingcompound, which is bound to the target protein. As another specificexample, either the first or second binding compound may generatemolecule that acts in conjunction with a proximity probe with aneffective proximity, producing a signal that correlates with thepresence of the target protein. Further, in another embodiment, bindingcompounds may have molecular tags that interact with one another withinan effective proximity to form a complex that generates a signal or canbe detected and measured in a manner that correlates with the presenceof the target protein. More specifically, the target protein or complexmay be p95 or a p95 complex.

“Binding moiety” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of binding to ananalyte. Binding moieties include, but are not limited to, antibodies,peptides, proteins, nucleic acids and organic molecules having amolecular weight of up to about 1000 Daltons and containing atomsselected from the group consisting of hydrogen, carbon, oxygen,nitrogen, sulfur and phosphorus. Preferably, binding moieties areantibodies.

“Cell lines” refers to cells that have been separated from theiroriginal tissue, clonally multiplied and/or maintained in culture. Asspecific examples, cell lines may be derived from each type of cancerand multiple different cell lines may be derived from samples of thesame type of cancer. Examples of different types of cell lines include,but are not limited to, breast cancer cell lines, such as MCF-7,MDA-MB-231, SK-BR-3, T-47D and ZR-75-1.

“Chemotherapeutic agent” means a chemical substance that is used totreat a condition, particularly cancer.

A “cleavable linkage,” as used herein, refers to a chemical linkinggroup that may be cleaved to release a detectable molecular tagconnected to a binding moiety with the cleavable linkage.

A “cleavage-inducing moiety,” or “cleaving agent,” as used herein, is agroup that produces an active species that is capable of cleaving acleavable linkage. Preferably, the active species is a chemical speciesthat exhibits short-lived activity so that its cleavage-inducing effectsare only in the proximity of the site of its generation.

A “cleaving probe,” as used herein, refers to a reagent that comprises acleavage-inducing moiety, as defined herein, and a binding compound suchas an antibody, a peptide, a peptide or non-peptide ligand for a cellsurface receptor, a protein, such as streptavidin, a small molecule,such as biotin, an oligonucleotide, an oligonucleotide analog, such as apeptide nucleic acid, a lectin or any other molecular entity that iscapable of binding to a target protein or molecule or stable molecularcomplex.

“Effective proximity,” as used herein, describes the distance betweentwo binding compounds that is sufficient to generate a detectablesignal, indicating the presence of the target molecule. For example, aproximity probe and a binding compound that are bound on p95 (or withanother analyte of interest) within an effective proximity will generatea detectable signal, indicating and/or quantifying the presence of p95and/or a p95 complex. Preferably, the effective proximity range for manydetection systems is less than 400 nM, preferably less than 300 nM,preferably less than 200 nM, preferably less than 100 nM, preferably,less than 50 nM.

“Epitope” refers to a site on the surface of a molecule, usually aprotein, to which an antibody molecule or other binding compound binds.Generally, a protein has several or many different epitopes, also calledantigenic determinants, and reacts with antibodies of differentspecificities. A preferred antigenic determinant is a phosphorylationsite of a protein. Preferred antigenic determinants are cryptic epitopesfound in the amino acid sequence of Her2 that are not accessible forbinding (e.g., by binding compounds) in the full length molecule butrather are revealed and accessible for binding in the truncated p95version of Her2.

“Exosome” refers to a membrane vesicle that has been released from acell membrane into an extracellular environment. Exosomes and othermembrane vesicles contain membrane-bound moieties, such as proteins, andthey may be used, for example, in assays to detect these moieties, suchas p95.

“Extracellular domain” refers to a portion of a molecule that liesoutside the membrane of a cell. An example of an extracellular domain,without limitation, would be the portion of a trans-membrane proteinthat lies outside the cell. More specifically, an example would be theextracellular domain of HER-2, which can be cleaved to generate a shedecto-domain and a truncated membrane-bound p95 protein.

“FFPE” shall refer to a formalin-fixed paraffin-embedded sample orsamples. Such samples are typically, for example, without limitation,used in an assay for proteins and receptor complexes in the form of thinsections, e.g. 3-10 μm thick, of fixed tissue mounted on a microscopeslide or equivalent surface. Such samples also typically undergo aconventional re-hydration procedure, and optionally, an antigenretrieval procedure as a part of, or preliminary to, assay measurements.

“Her-2”, “ErbB2”, “c-Erb-B2”, “HER2”, “Her2” and “neu” are usedinterchangeably herein and refer to native Her-2, and allelic variantsthereof, as described, for example, in Semba et al., 1985, P.N.A.S. USA82:6497-650 and Yamamoto et al., 1986, Nature 319:230-234 and Genbankaccession number X03363. Unless indicated otherwise, the terms “Her-2”,“ErbB2”, “c-Erb-B2”, “HER2” and “Her2” when used herein refer to thehuman protein. The gene encoding Her2 is referred to herein as “erbB2.”

“Her-2-acting agent,” as used herein, refers to a compound that caninhibit a biological activity of Her-2 or a Her-2 expressing cell or aHer-2 positive cancer cell. Such biological activities include, but arenot limited to, dimerization, autophosphorylation, phosphorylation ofanother receptor, signal transduction and the like. Biologicalactivities can include, without limitation, cell survival and cellproliferation and inhibition of such activities by a Her-2 acting agentcould be direct or indirect cell killing (eg, ADCC), disruption ofprotein complexes or complex formation, modulation of proteintrafficking or enzyme inhibition. Biological activities can also includepatient response as set forth in this application. ExemplaryHer-2-acting agents include, but are not limited to BIBW 2992, HKI-272,4D5, pertuzumab, trastuzumab, Herceptin-DM-1, AEE-788 and lapatinib.

“High” refers to a measure that is greater than a standard such as apredetermined measure or a subgroup measure or that is relativelygreater than another subgroup measure. For example, high Her-2 or p95may refer to a measure that is equal to or greater than a predeterminedmeasure, such as a predetermined cutoff. High Her-2 or p95 may alsorefer to a measure of Her-2 or p95 wherein a high Her-2 or p95 subgrouphas relatively greater levels of Her-2 or p95 than another subgroup. Forexample, without limitation, according to the present specification, twodistinct patient subgroups can be created by dividing samples around amathematically determined point, such as, without limitation, a median,thus creating a subgroup whose measure is high (i.e., higher than themedian) and another subgroup whose measure is low. Her-2 or p95 can bemeasured by any method known to one skilled in the art such as, forexample, without limitation, using VeraTag or using any standardimmunohistochemical (IHC) method such as HercepTest®.

“Likely to,” as used herein, refers to an increased probability that anitem, object, thing or person will occur. Thus, in one example, asubject that is likely to respond to treatment with trastuzumab has anincreased probability of responding to treatment with trastuzumabrelative to a reference subject or group of subjects.

“Long,” as used herein, refers to a time measure that is greater than apredetermined measure or a subgroup measure that is relatively longerthan another subgroup measure. For example, with respect to a patient'slongevity, a long time progression refers to time progression that islonger than expected. Whether a time progression is long or not may bedetermined according to any method available to one skilled in the art.

“Low” is a term that refers to a measure that is less than a standardsuch as a predetermined measure or a subgroup measure that is relativelyless than another subgroup measure. For example, low Her-2 or p95 maymean a method that is less than a predetermined measure, such as apredetermined cutoff. Low Her-2 or p95 may also mean a measure wherein alow Her-2 or p95 subgroup is relatively lower than another subgroup. Forexample, without limitation, according to the present specification, twodistinct patient subgroups can be created by dividing samples around amathematically determined point, such as, without limitation, a median,thus creating a group whose measure is low (i.e., less than the median)with respect to another group whose measure is high (i.e., greater thanthe median). Her-2 or p95 can be measured by any method known to oneskilled in the art such as, for example, without limitation, using theVeraTag method or using any standard immunohistochemical (IHC) methodsuch as HercepTest®.

A “molecular tag,” as used herein, refers to a molecule that can bemeasured directly or indirectly, can be distinguished from othermolecules based on one or more physical, chemical or optical differencesamong the molecules being separated, including but not limited to,electrophoretic mobility, molecular weight, shape, solubility, pKa,hydrophobicity, charge, charge/mass ratio, polarity or the like. In oneembodiment, molecular tags in a plurality or set differ inelectrophoretic mobility and optical detection characteristics and canbe separated by electrophoresis. In another embodiment, molecular tagsin a plurality or set may differ in molecular weight, shape, solubility,pKa, hydrophobicity, charge, polarity and can be separated by normalphase or reverse phase HPLC, ion exchange HPLC, capillaryelectrochromatography, mass spectroscopy, gas phase chromatography or alike technique.

Measurement of molecular tags may also involve using secondary molecularinteractions, with or without further modification, to detect, enhanceor amplify a measurable signal that acts as a correlative for thepresence and/or quantity of an analyte, such as p95 or a p95 complex. Inone embodiment, a set of two or more molecular tags may interact withinan effective proximity to produce a measurable signal. As moleculartags, a measurable signal may be generated, for example, by detection oftwo complementary nucleic acid sequences which will hybridize when thecomplementary sequences are within an effective proximity. Otherexamples that either generate a measurable signal or that can bemeasured using detection methods know in the art include, but are notlimited to, FRET, BRET, BiFC, LCI and QPCR.

“Optimal cutoff” as used herein, refers to the value of a predeterminedmeasure on subjects exhibiting certain attributes that allow the bestdiscrimination between two or more categories of an attribute. Forexample, an optimal cutoff that allows one to best discriminate betweentwo categories such as high p95 expression and low p95 expression fordetermining overall survival would be useful. Optimal cutoffs may beused to separate the subjects with values lower than or higher than theoptimal cutoff to optimize the prediction model.

“Overall survival” or “OS” refers to a time as measured from the startof treatment to death or censor. Censoring may come from a study end orchange in treatment. Overall survival can refer to a probability as, forexample, a probability when represented in a Kaplan-Meier plot of beingalive at a particular time, that time being the time between the startof the treatment to death or censor.

“p95” refers to an N-terminally truncated, C-terminal portion of HER-2.“p95” has also been referred to as “truncated ErbB2 receptor”,“p95^(ErbB2)”, “p95HER2”, and more generally as “NH₂-terminallytruncated HER-2/neu” and “HER2 C-terminal fragments” to reflect the factthat “p95” represents a family of truncated HER2 proteins similar, butnot identical in size to that originally identified as having anapparent molecular weight of 95 kiloDaltons. p95 is thought to beproduced by at least two distinct mechanisms. p95 may result from theproteolytic cleavage of full-length HER-2. p95 may also result from analternative translational start downstream from the canonical firstmethionine including but not limited to M611 and M687

“p95 complex” refers to a complex of proteins at least one member ofwhich is p95. Examples, without limitation, of possible p95 complexesinclude p95 homodimers, as well as heterodimers comprised of p95 andfull-length Her2 and also other members of the epidermal growth factorreceptor family including Her1, Her3 and Her4.

A “proximity probe,” as used herein, refers to a reagent that comprisesa moiety capable of acting within effective proximity to a molecular tagon a binding compound to generate a detectable signal and an antibody, apeptide, a peptide or non-peptide ligand for a cell surface receptor, aprotein, such as streptavidin, a small molecule, such as biotin, anoligonucleotide, an oligonucleotide analog, such as a peptide nucleicacid, a lectin or any other molecular entity that is capable ofspecifically binding to a target protein or molecule or stable complex.For example, a proximity probe comprised of a p95-targeted antibody witha molecular tag may be capable of binding to p95 within an effectiveproximity to one or more p95 binding compounds, or a binding compound ofanother protein of interest, that has one or more molecular tagsattached. In one embodiment, a proximity probe comprises a bindingmolecule and a first nucleic acid and a binding molecule comprises anantibody and a second nucleic acid, wherein the first and second nucleicacids are complementary to each other and each is a predetermined lengthso that when the nucleic acids are within an effective proximity of oneanother, they hybridize. Hybridization may be measured by any methodknown to one skilled in the art. For example, fluorophores may beattached to the nucleic acids as indicators of hybridization. In apreferred embodiment, hybridization is measured with a nucleic acidamplification method such as, for example, without limitation, therolling circle amplification method (see, for example, Lizardi et al.,(1998) Nat Genet. 19: 225-232).

“RECIST” shall mean “Response Evaluation Criteria in Solid Tumours” andis a set of published rules that define when cancer patients improve(“respond”), stay the same (“stable”) or worsen (“progression”) duringtreatments. Response as defined by RECIST criteria have been published,for example, at Journal of the National Cancer Institute, Vol. 92, No.3, Feb. 2, 2000 and RECIST criteria may include other similar publisheddefinitions and rule sets.

“Respond” to treatment, and other forms of this verb, as used herein,refer to the reaction of a subject to treatment with an agent. As anexample, a subject responds to treatment if growth of a tumor in thesubject is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore. In another example, a subject responds to treatment if a tumor inthe subject shrinks by about 5%, 10%, 20%, 30%, 40%, 50% or more asdetermined by any appropriate measure, e.g., by mass or volume. Inanother example, a subject responds to treatment with a Her2-actingagent if the subject experiences a life expectancy extended by about 5%,10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted ifno treatment is administered. In another example, a subject responds totreatment with an agent if the subject has an overall survival orincreased time to progression. Several methods may be used to determineif a patient responds to a treatment including the RECIST criteria, asset forth herein.

“Sample” or “tissue sample” or “patient sample” or “patient cell ortissue sample” or “specimen” each refer to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample may be solid tissue as from a fresh tissue, frozen and/orpreserved organ or tissue or biopsy or aspirate; blood or any bloodconstituents, bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid or cells from any time ingestation or development of the subject. The tissue sample may containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. Cells may be fixed in a conventional manner,such as in an FFPE manner.

“Short,” as used herein, refers to a time measure that is shorter than astandard such as a predetermined measure or a subgroup measure that isrelatively shorter than another subgroup measure. For example, withrespect to a patient's longevity, a short time progression refers totime progression that is shorter than predicted. Whether a timeprogression is short or not may be determined according to any methodavailable to one skilled in the art.

“Significant event,” as used herein, shall refer to an event in apatient's disease that is important as determined by one skilled in theart. Examples of significant events include, for example, withoutlimitation, primary diagnosis, death, recurrence, the determination thata patient's disease is metastatic, relapse of a patient's disease or theprogression of a patient's disease from any one of the above notedstages to another. A significant event may be any important event usedto assess OS, TTP and/or using the RECIST or other response criteria, asdetermined by one skilled in the art.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouseor sheep) and a primate (e.g., a monkey, such as a cynomolgus monkey,gorilla, chimpanzee or a human).

“Targeted therapy” refers to therapeutic treatment that attempts toidentify and treat specific cells involved in disease without harming oraltering normal cells. Targeted therapeutics may be comprised of, butnot limited to, small molecules, such as lapatinib, imatinib (such asIressa®) and gefitinib (GLEEVEC®), monoclonal antibodies, such astrastuzumab or nucleic acids, such as siRNAs used to block expression ofgene products involved in disease processes. Targeted therapies areuseful in the treatment of many disease processes, such as cancer. Asused herein, “time course” shall refer to the amount of time between aninitial event and a subsequent event. For example, with respect to apatient's cancer, time course may relate to a patient's disease and maybe measured by gauging significant events in the course of the disease,wherein the first event may be diagnosis and the subsequent event may bemetastasis, for example.

“Time to progression” or “TTP” refers to a time as measured from thestart of the treatment to progression or a cancer or censor. Censoringmay come from a study end or from a change in treatment. Time toprogression can also be represented as a probability as, for example, ina Kaplan-Meier plot where time to progression may represent theprobability of being progression free over a particular time, that timebeing the time between the start of the treatment to progression orcensor.

“Treatment,” and other forms of this word refer to the administration ofan agent to impede a disease, such as the growth of a cancer, to cause acancer to shrink by weight or volume, to extend the expected survivaltime of the subject and/or time to progression of the tumor or the like.Treatment may also refer to any course which one skilled, for example, atreating physician, deems expedient.

“Tumor lysate” refers to the solution produced when the cell membranesof tumors are disrupted, whether by physical or chemical methods. Tumorlysates typically contain representative components of the cell,including but not limited to, protein markers, enzymes, nucleic acidsand complexes of proteins and other molecules that can subsequently bemeasured in various assays.

The term “VeraTag” refers to single and multiplexed and multi-labelassays, materials, methods and techniques for performing and utilizingsuch assays, including but not limited to reagents, analyticalprocedures and software related to those assays. The terms VeraTag, vTagand eTag shall be used interchangeably.

In a first aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or amount of p95 or p95 complex in asample, the method comprising providing a sample and determining thepresence and/or quantity of p95 or p95 complex in the sample. In apreferred embodiment, the sample is a biological sample. In a preferredembodiment, the sample is a tissue sample. In a preferred embodiment,the sample is a fixed sample, a frozen sample or a lysate. In apreferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample contains exosomes and/or othervesicles. In a preferred embodiment, the sample comprises cell lines. Ina preferred embodiment, the measurement may be quantitative across awide dynamic range.

In a second aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or quantity of p95 in a sample, themethod comprising mixing a sample with a binding compound anddetermining the presence and/or quantity of binding compound bound top95. In a preferred embodiment, the binding compound is capable ofspecifically binding p95. In a preferred embodiment, the bindingcompound comprises an antibody. In a preferred embodiment, the antibodywas raised against one of the peptides having SEQ ID NOs 1-7. In certainembodiments, the antibody is or comprises one of the antibodies producedby hybridoma cell lines deposited with the ATCC having accession numberPTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1). In oneembodiment, the antibody is p95.D9.1. In a preferred embodiment, thesample is a biological sample. In a preferred embodiment, the sample isa tissue sample. In a preferred embodiment, the sample is a fixedsample, a frozen sample or a lysate. In a preferred embodiment, thesample is a tumor sample. In a preferred embodiment, the sample is afrozen tumor tissue sample. In a preferred embodiment, the samplecomprises a tumor lysate. In a preferred embodiment, the samplecomprises a breast cancer sample. In a preferred embodiment, the sampleis an FFPE sample. In a preferred embodiment, the sample is a blood,plasma or lymph sample. In a preferred embodiment, the blood or plasmasample contains circulating tumor cells. In a preferred embodiment, thesample contains exosomes and/or other vesicles. In a preferredembodiment, the sample comprises cell lines. In a preferred embodiment,the measurement may be quantitative across a wide dynamic range.

In a preferred embodiment, determining the presence and/or quantity ofbinding compound bound to p95 further comprises providing a secondbinding compound, the second binding compound being able to specificallybind the binding compound bound to p95 and determining the presenceand/or quantity of the second binding compound as correlative of thepresence and/or quantity of the binding compound bound to p95. In apreferred embodiment, the second binding compound is an antibody.

The use of a second binding compound that is capable of specificallybinding the first binding compound and has one or more molecular tagsmay have practical advantages. For example, multiple p95-specific firstbinding compounds may be tested using a single second binding compoundto which is attached one or more molecular tags, abrogating the need forattaching molecular tags to each of the multiple p95-specific firstbinding compounds. In a preferred embodiment, the first binding compoundis a mouse antibody and the second binding compound is an anti-mouseantibody raised in a non-mouse species (e.g., goat anti-mouseantibodies) to which cleavable molecular tags have been attached.

Second binding compounds are typically labeled with probes useful fordetection. Detection systems commonly used for detecting second bindingcompounds include but are not limited to cleavable molecular tags, asdescribed herein; radiolabels (i.e., radioisotopes such as 1-125);enzymes that convert a chemical into a measurable colorimetric,fluorescent or electrochemical signal (e.g., peroxidases) andfluorescent proteins (e.g., green fluorescent protein and its manyderivatives). One of the most commonly used detection systems, forexample, for immunohistochemistry, is to conjugate horseradishperoxidase (HRP) to an antibody or other binding compound. A substratecan then be oxidized by HRP, yielding a product detectable by aspectrophotometric method. Substrates for HRP include both chromogenicsubstrates (e.g., 3,3′,5,5′-tetramethylbenzidine [TMB] or3,3′-diaminobenzidine [DAB]), which yield colored products andchemiluminescent substrates (e.g., enhanced luminol chemiluminescence[ECL]), which yield light. Immunohistochemistry detection methods usingsecondary binding compounds and peroxidases typically involve eithertagging the primary binding compound with a small molecule that can bebound with a second binding compound to which a peroxidase has beenconjugated (e.g., a streptavidin/biotin system) or using a secondaryantibody that has been conjugated with peroxidase targeted to the firstantibody (e.g., a goat-anti-mouse antibody). Substrate is then addedunder conditions that will allow the conversion to product in arelatively quantitative manner and spectrophotometric methods are thenused to detect the product.

The antibody can be monoclonal, polyclonal or recombinant and can beprepared by techniques that are well known in the art. Antibodies mayinclude a complete immunoglobulin or fragment thereof, whichimmunoglobulins include the various classes and isotypes, such as IgA,IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′ and the like. Antibodies may also besingle-chain antibodies, chimeric antibodies, humanized antibodies orany other antibody derivative known to one of skill in the art thatretains binding activity that is specific for a particular binding site.In addition, aggregates, polymers and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity is maintained.

To facilitate the development of methods to measure p95 in biologicalsamples, p95-specific monoclonal antibodies were created. Mice wereimmunized against peptides from p95 and standard methods as set forthfurther herein and as known to one skilled in the art were used tocreate hybridomas. Many methods are known for the creation andproduction of monoclonal antibodies, for example, the hybridoma methodas first described by Koehler et al. (1975) Nature 256:495-497 or othermethods described in the literature (see Goding, J W (1980) J. Immunol.Methods 34:285-308; Harlow E and Lane D (1988) in Antibodies: ALaboratory Manual, Chapter 6; Kennett R H et al. (1980) MonoclonalAntibodies, Plenum Press; Zola H (1987) Monoclonal Antibodies: A Manualof Techniques, CRC Press).

In one embodiment, the method of creating hybridomas begins withimmunizing a host animal, such as a mouse, to elicit the production oflymphocytes that produce antibodies targeted to the peptide orprotein(s) of interest. Lymphocytes may also be immunized in vitro. Theantigen used may be a peptide, a protein or a cell displaying theantigen on the cell surface. Lymphocytes are collected then fused bychemical (e.g., with PEG) or electrical (e.g., by electrofusion) methodswith myeloma cells to form hybridoma cells, typically under conditionsthat prevent the growth and/or survival of the parent myeloma cells.Fused cells are allowed to grow because they contain enzymes thatfacilitate survival in the culture medium. In a preferred embodiment,the culture medium contains hypoxanthine, aminopterin and thymidine (HATmedium), which prevents the growth of cells lacking hypoxanthine quininephosphoribosyl transferase (HPRT). The HPRT is supplied to the fusedcell by the lymphocyte partner, allowing survival of the hybridoma butpreventing survival of the parent myeloma cells, which lack HPRT.

Culture media in which hybridomas are grown (i.e., conditioned media)are typically assayed for the production of monoclonal antibodiesdirected against the antigen using a variety of techniques (see Voller,et al. (1978) J. Clin. Pathol. 31:507-520), including but not limitedto, immunoprecipitation or an in vitro binding assay such asenzyme-linked immunosorbant assay (ELISA; see Engvall E (1977) inBiomedical Applications of Immobilized Enzymes and Proteins, edited byTMS Chang, 2:87-96, Plenum Press), radioimmunoassay (RIA; see Sonksen PH (1974) Brit. Med. Bull. 30:1-103), Western blots or flow cytometry.Conditioned media from the hybridomas were profiled in a series ofassays including ELISA (FIG. 1), Western blot (FIG. 2) and flowcytometry (FIG. 3). In preferred embodiments, studies using both nativeand permeabilized and fixed cells are performed to identify antibodiesthat may perform well in applications that use fixed cells or tissues,such as immunohistochemistry (IHC). Clones of interest may be subclonedby limiting dilution or single cell flow cytometry.

As will be known to those skilled in the art, monoclonal antibodiessecreted by hybridoma clones (or subclones) can be purified usingconventional purification procedures such as, but not limited to,dialysis, affinity chromatography, gel electrophoresis or proteinA-sepharose (or protein L-agarose) chromatography.

Many methods and reagents are commonly used to prepare biologicalsamples for analysis. Several methods are outlined or referenced hereinand many others are known to those skilled in the art. Samplescontaining p95 suitable for use as biomarkers may come from a widevariety of sources, including cell cultures, animal or plant tissues,patient biopsies, blood or the like. Preferably, samples are humanpatient samples. Samples are prepared for assays of the invention usingconventional techniques, which may depend on the source from which asample is taken. For biopsies and medical specimens, guidance isprovided in the following references: Bancroft J D & Stevens A, eds.1977, Theory and Practice of Histological Techniques, ChurchillLivingstone, Edinburgh; Pearse, 1980, Histochemistry. Theory andapplied. 4^(th) ed., Churchill Livingstone, Edinburgh.

Examples of patient tissue samples that may be used include, but are notlimited to, tissues of breast, prostate, ovary, colon, lung,endometrium, stomach, salivary gland or pancreas. The tissue sample canbe obtained by a variety of procedures including surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In oneembodiment, the biological sample may be cells cultured in vitro andcollected by centrifugation as a cell pellet. In one embodiment, thesamples may be patient blood samples or specific blood cell types orsubsets of blood cell types (e.g., buffy coats). In one embodiment, thebiological sample may be exosomes or samples containing exosomes.Exosomes are small (30-200 nm) vesicles that can be secreted by mostcell types, including tumor cells (see Mignot et al (2006) J. Cell. Mol.Med. 10:376-388), in vivo and in vitro. Tumor-derived exosomes arethought to play a role in the ability of tumors to evade the immunesystem and have potential for both diagnostic and therapeuticapplications (see Taylor and Black (1985) J. Natl. Cancer Inst.74:859-867) and are therefore biological samples of interest.

In a preferred embodiment, the sample is a tumor sample. Examples oftypes of tumor samples include cancers such as, without limitation,carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed typecancers. In one embodiment, the cancer is a bone cancer, for example,Ewing's sarcoma, osteosarcoma and rhabdomyosarcoma and other soft-tissuesarcomas. In another embodiment, the cancer is a brain tumor, forexample, oligodendroglioma, ependymoma, menengioma, lymphoma orschwannoma ormedulloblastoma. In another embodiment, the cancer isbreast cancer. In another embodiment, the cancer is an endocrine systemcancer, for example, adrenal, pancreatic, parathyroid, pituitary andthyroid cancers. In another embodiment, the cancer is a gastrointestinalcancer, for example, anal, colorectal, esophogeal, gallbladder, gastric,liver, pancreatic and small intestine cancers. In another embodiment,the cancer is a gynecological cancer, for example, cervical,endometrial, uterine, fallopian tube, gestational trophoblastic disease,choriocarcinoma, ovarian, vaginal and vulvar cancers. In anotherembodiment, the cancer is a head and neck cancer, for example,laryngeal, oropharyngeal, parathryroid or thyroid cancer. In anotherembodiment, the cancer is a leukemic cancer, for example, acutelymphocytic leukemia, acute myelogenous leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia or amyeloproliferative disorder. In another embodiment, the cancer is a lungcancer, for example, a mesothelioma or non-small cell lung cancer. Inanother embodiment, the cancer is a lymphoma, cutaneous T cell lymphoma,Hodgkin's disease or non-Hodgkin's disease. In another embodiment, thecancer is metastatic cancer. In another embodiment, the cancer is amyeloma, for example, a multiple myeloma. In another embodiment, thecancer is penile cancer. In another embodiment, the cancer is prostatecancer. In another embodiment, the cancer is testicular cancer. Inanother embodiment, the cancer is thyroid cancer, for example,papillary, follicular, medullary or anaplastic or undifferentiatedthyroid carcinoma. In another embodiment, the cancer is urinary tractcancers, for example, bladder, kidney or urethral cancers.

Methods for preparing cells cultured in vitro as fresh, frozen or fixedsamples are known to those with skill in the art and exemplary methodsare described herein. In one embodiment, assays of the invention arecarried out on tissue samples that have been fixed and embedded inparaffin and a step of deparaffination may be carried out. A tissuesample may be fixed (i.e., preserved) by conventional methodology. See,e.g., Lee G. Luna, H T (ASCP) Ed., 1960, Manual of Histological StainingMethod of the Armed Forces Institute of Pathology 3^(rd) edition, TheBlakston Division McGraw-Hill Book Company, New York; Ulreka V. Mikel,Ed., 1994, The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology, Armed Forces Institute of Pathology,American Registry of Pathology, Washington, D.C. One of skill in the artwill appreciate that the choice of a fixative is determined by thepurpose for which the tissue is to be histologically stained orotherwise analyzed. One of skill in the art will also appreciate thatthe length of fixation depends upon the size of the tissue sample andthe fixative used.

Generally, a tissue sample is first fixed and is then dehydrated throughan ascending series of alcohols, infiltrated and embedded with paraffinor other sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology according to conventionaltechniques or as described herein. Once the tissue sample is embedded,the sample may be sectioned by a microtome according to conventionaltechniques. Sections may have a thickness in a range from about threemicrons to about twelve microns, and preferably, a thickness in a rangeof from about 5 microns to about 10 microns. In one embodiment, asection may have an area of from about 10 mm² to about 1 cm². Once cut,the sections may be attached to slides by several standard methods.Examples of slide adhesives include, but are not limited to, silane,gelatin and poly-L-lysine. Paraffin-embedded sections may be attached topositively charged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated prior to detection ofbiomarkers. Tissue sections may be deparaffinized by severalconventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used according toconventional techniques described by the references provided herein.Alternatively, commercially available deparaffinizing non-organic agentssuch as Hemo-De® (CMS, Houston, Tex.) may be used.

Cell lysates of mammalian tissue culture cells or fresh or frozentissues may be prepared by conventional cell lysis techniques (e.g.,0.14 M NaCl, 1.5 mM MgCl₂, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40,and protease and/or phosphatase inhibitors as required). For freshmammalian tissues, sample preparation may also include a tissuedisaggregation step, such as crushing, mincing, grinding or sonication.

Cell lysates were prepared and tested in Western blots against Ab8 orCB11, anti-Her2 antibodies that bind an intracellular epitope of Her2and are thus capable of detecting both full length Her2 and p95. Theresults confirm that both the SKBR3 and MCF7 cell lines transfected withthe p95 expression vectors express p95 and that transfected full lengthHer2 is also expressed in MCF7 cells. The SKBR3 cell line expressesendogenous high levels of Her2, as well as some p95, which is expectedbecause SKBR3 is known to shed Her2-ECD (see Zabrecky et al. (1991) J.Biol. Chem. 266:1716-1720).

Six cell lines were tested using the putative p95-specific monoclonalantibodies: MCF7; MCF7 transfected with either full length Her2, p95 orCTF; SKBR3 and SKBR3 transfected with HER2 carboxy terminal fragments(CTF). CTF and p95 are used interchangeably to describe the family oftruncated HER2 with PAGE apparent molecular weights similar to 95kiloDaltons (see definition of p95 and Anido et al. (2006) EMBO J.12:3234-3244). The results are shown in FIG. 6. Two monoclonalantibodies were shown to be specific for p95, D8.2 and D9.1.

The ability of the p95-specific antibody D9.1 to detect p95 in tumorswas tested in tumor samples, which were selected to have a highprobability of containing p95. Her2-positive tumor samples from patientswith node-positive status (both factors correlating with p95-positivity)were obtained as matched fresh-frozen and formalin-fixed,paraffin-embedded samples. Western blots were prepared with lysates fromthe fresh-frozen material and probed with CB11, a commercially-availableantibody targeted to the intracellular domain of Her2. The results,shown in FIG. 7 a, allowed assignation of p95-positive status to 7 ofthe 12 tumor samples. The FFPE samples of all 12 tumors were then testedin the VeraTag assay using the D9.1 antibody as described in Example 4(also see, for example, FIGS. 7 b and 7 c). Tumor samples areheterogeneous in nature, so some differences in the p95 levels offresh-frozen and FFPE samples were expected.

Isotype controls are typically performed to eliminate the possibilitythat the binding results are due to the particular isotype of theantibody rather than the individual antibody. Additionally, one skilledin the art will appreciate that any signal “noise” seen in the isotypecontrols can be subtracted from the total signal, potentially yielding amore refined result. When an isotype control experiment was performed(see FIGS. 8 a and 8 b) and the control result subtracted from the testresult with D9.1, the difference between the median p95-positive andp95-negative populations was retained and significant (approximately4-fold different with a dynamic range of approximately 10-fold; see FIG.8 c).

In a third aspect, the invention is drawn to a method of measuringand/or quantifying the presence and/or quantity of p95 or a p95 complexin a sample, the method comprising: mixing (i) a sample that may containp95 or a p95 complex; (ii) a proximity probe that is capable of bindingp95 or an analyte which binds p95 or a p95 complex, the proximity probehaving an effective proximity and (iii) at least one binding compound,the at least one binding compound being capable of binding p95, or ananalyte which binds p95. and having one or more molecular probesattached, wherein binding of the proximity probe and binding compoundwithin the effective proximity produces a signal from the molecularprobes that correlates with the presence and/or quantity of p95 or p95complex. In a preferred embodiment, the proximity probe and/or bindingcompound is capable of specifically binding p95. In a preferredembodiment, the proximity probe and/or binding compound furthercomprises an antibody. In a preferred embodiment, the proximity probeand/or the binding compound further comprises an antibody, and eachantibody binds to a specific epitope on p95. In a preferred embodiment,the antibody was raised against one of the peptides having SEQ ID NOs1-7. In certain embodiments, the antibody is or comprises one of theantibodies produced by hybridoma cell lines deposited with the ATCChaving accession number PTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) andPTA-9740 (p95.D9.1). In one embodiment, the antibody is p95.D9.1.

In a preferred embodiment, the proximity probe comprises an antibody anda first nucleic acid and the binding compound comprises an antibody anda second nucleic acid, wherein the first and the second nucleic acidsare complementary to each other and able to hybridize to determine theeffective proximity and produce the signal through hybridization.Hybridization may be quantified by any method known to one skilled inthe art such as, for example, measuring molecular tags attached to thenucleic acid molecules or measuring hybridization with any method knownto one skilled in the art. In a preferred embodiment, hybridization ismeasured through a nucleic acid amplification method such as, forexample, the rolling circle amplification method. In a preferredembodiment, the antibody was raised against one of the peptides havingSEQ ID NOs 1-7. In certain embodiments, the antibody is or comprises oneof the antibodies produced by hybridoma cell lines deposited with theATCC having accession number PTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2)and PTA-9740 (p95.D9.1). In one embodiment, the antibody is p95.D9.1. Ina preferred embodiment, the sample is a biological sample. In apreferred embodiment, the sample is a tissue sample. In a preferredembodiment, the sample is a fixed sample, a frozen sample or a lysate.In a preferred embodiment, the sample is a tumor sample. In a preferredembodiment, the sample is a frozen tumor tissue sample. In a preferredembodiment, the sample comprises a tumor lysate. In a preferredembodiment, the sample comprises a breast cancer sample. In a preferredembodiment, the sample is an FFPE sample. In a preferred embodiment, thesample is a blood, plasma or lymph sample. In a preferred embodiment,the blood or plasma sample contains circulating tumor cells. In apreferred embodiment, the sample contains exosomes and/or othervesicles. In a preferred embodiment, the sample comprises cell lines. Ina preferred embodiment, the measurement may be quantitative across awide dynamic range. Examples of proximity probes and binding compounds,as set forth herein, can be found, for example, in United States patentapplications 7,306,904; 7,320,860 and 7,351,528, each of which isincorporated by reference herein, including any drawings.

Proximity assays are increasingly useful for the understanding of thebiological role of molecular complexes, as well as in the study ofbiomarkers. For example, binding compounds that specifically bind p95 ora p95 complex can be coupled with many different detection systems tomeasure the presence and/or quantity of p95 or a p95 complex. Any methodknown to one of skill in the art to be useful for determining an amountof p95 or a p95 complex can be used in accordance with the presentinvention. Such methods include but are not limited to Foersterresonance energy transfer (FRET), bioluminescence resonance energytransfer (BRET), biomolecular fluorescence complementation, proximityligation assay (PLA), scintillation proximity assays (SPA) and rollingcircle amplification (RCA) or any other method for detecting nucleicacid duplexes formed by the proximity of a binding probe and a proximityprobe with complementary strands of nucleic acids.

In conducting the methods of the invention, a combination of the assaycomponents is made, including the sample being tested, the bindingcompounds and optionally the proximity probe. Generally, assaycomponents may be combined in any order. In certain applications,however, the order of addition may be relevant. For example, one maywish to monitor competitive binding, such as in a quantitative assay. Orone may wish to monitor the stability of an assembled complex. In suchapplications, reactions may be assembled in stages.

The amounts of each reagent can generally be determined empirically. Theamount of sample used in an assay will be determined by the predictednumber of target complexes present and the means of separation anddetection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the proximity probe can be providedin molar excess relative to the expected amount of the target moleculesin the sample, generally at a molar excess of at least about 1.5, moredesirably about 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding compound or proximity probe and the expected number oftarget molecules present on a single cell.

The assay mixture can be combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultured), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° to 70° C., usually from about 15° to 45° C., moreusually about 25° to 37° C.

In a preferred embodiment, the proximity probe comprises a proximityprobe that has a cleavage-inducing moiety and the at least one bindingcompound has one or more molecular tags attached to the binding compoundby a cleavable linkage, wherein the cleavable linkage may be cleavedwithin the effective proximity producing a signal that correlates withthe presence and/or quantity of p95. In a preferred embodiment, thebinding compound and/or the proximity probe further comprises anantibody, and each antibody binds to a specific epitope on p95 or ananalyte that binds p95. In a preferred embodiment, the antibody wasraised against one of the peptides having SEQ ID NOs 1-7. In certainembodiments, the antibody is or comprises one of the antibodies producedby hybridoma cell lines deposited with the ATCC having accession numberPTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1). In oneembodiment, the antibody is p95.D9.1. In a preferred embodiment, thesample is a biological sample. In a preferred embodiment, the sample isa tissue sample. In a preferred embodiment, the sample is a fixedsample, a frozen sample or a lysate. In a preferred embodiment, thesample is a tumor sample. In a preferred embodiment, the sample is afrozen tumor tissue sample. In a preferred embodiment, the samplecomprises a tumor lysate. In a preferred embodiment, the samplecomprises a breast cancer sample. In a preferred embodiment, the sampleis an FFPE sample. In a preferred embodiment, the sample is a blood,plasma or lymph sample. In a preferred embodiment, the blood or plasmasample contains circulating tumor cells. In a preferred embodiment, thesample contains exosomes and/or other vesicles. In a preferredembodiment, the sample comprises cell lines. In a preferred embodiment,the measurement may be quantitative across a wide dynamic range.

Many advantages are provided by measuring p95 or a p95 complex usingreleasable molecular tags, including separation of released moleculartags from an assay mixture providing greatly reduced background and asignificant gain in sensitivity and separation and detection providing aconvenient multiplexing capability so that multiple receptor complexcomponents may be readily measured simultaneously in the same assay.Assays employing such tags can have a variety of forms and are disclosedin the following references: U.S. Pat. Nos. 7,105,308; 6,627,400;7,402,397; 7,402,398 and 7,402,399, as well as International PatentPublication No. WO 2004/011900, each of which is incorporated herein byreference in its entirety. A wide variety of separation techniques maybe employed that can distinguish molecules based on one or morephysical, chemical or optical differences among molecules beingseparated including electrophoretic mobility, molecular weight, shape,solubility, pKa, hydrophobicity, charge, charge/mass ratio or polarity.In one embodiment, molecular tags in a plurality or set differ inelectrophoretic mobility and optical detection characteristics and areseparated by electrophoresis. In another embodiment, molecular tags in aplurality or set may differ in molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity and are separated by normal phase orreverse phase HPLC, ion exchange HPLC, capillary electrochromatography,mass spectroscopy or gas phase chromatography.

Sets of molecular tags are provided that can be separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the presence and/or amounts of p95.Molecular tags within a set may be chemically diverse; however, forconvenience, sets of molecular tags are usually chemically related. Forexample, they may all be peptides or they may consist of differentcombinations of the same basic building blocks or monomers or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics. The number ofmolecular tags in a plurality may vary depending on several factorsincluding the mode of separation employed, the labels used on themolecular tags for detection, the sensitivity of the binding moietiesand the efficiency with which the cleavable linkages are cleaved.

Measurements made directly on tissue samples may be normalized byincluding measurements on cellular or tissue targets that arerepresentative of the total cell number in the sample and/or the numbersof particular subtypes of cells in the sample (see, for example, UnitedStates Provisional Application No. 61/015,608 which is incorporated byreference herein, including any drawings). The additional measurementmay be preferred, or even necessary, because of the cellular and tissueheterogeneity in patient samples, particularly tumor samples, which maycomprise substantial fractions of normal cells.

In one embodiment, a binding compound can be represented by thefollowing formula:

B-(L-E)_(k)

wherein B is binding moiety; L is a cleavable linkage and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g. 100 to 500 or k is greater than several hundred to as many asseveral thousand, e.g. 500 to 5000. Usually each of the plurality ofdifferent types of binding compounds has a different molecular tag, E.Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

Preferably, B is an antibody that specifically binds to a target, suchas p95. Antibodies specific for p95 epitopes are provided in theexamples set forth herein. Antibody compositions may be readily formedfrom a wide variety of commercially available antibodies, eithermonoclonal or polyclonal or by methods disclosed herein.

Cleavable linkage, L, can be virtually any chemical linking group thatmay be cleaved under conditions that do not degrade the structure oraffect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages within an effective proximity of the proximity probe arecleaved. Typically, such an agent must be activated by making a physicalor chemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toaffect cleavage.

In a non-homogeneous format, because specifically-bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages andpeptide linkages cleavable by specific proteases. References describingmany such linkages include Greene and Wuts, 1991, Protective Groups inOrganic Synthesis, Second Edition, John Wiley & Sons, New York;Hermanson, 1996, Bioconjugate Techniques, Academic Press, New York; andU.S. Pat. No. 5,565,324, each of which is incorporated by referenceherein.

Molecular tag, E, in the present invention may comprise an electrophorictag as described in the following references when separation ofpluralities of molecular tags are carried out by gas chromatography ormass spectrometry: See, e.g., Zhang et al., 2002, Bioconjugate Chem.13:1002-1012; Giese, 1983, Anal. Chem. 2:165-168; and U.S. Pat. Nos.4,650,750; 5,360,819; 5,516,931; and 5,602,273, each of which is herebyincorporated by reference in its entirety.

Molecular tag, E, is preferably a water-soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. In one embodiment, E has a molecularweight in the range of from about 50 to about 2500 Daltons, morepreferably, from about 50 to about 1500 Daltons. E may comprise adetection group for generating an electrochemical, fluorescent orchromogenic signal. In embodiments employing detection by mass, E maynot have a separate moiety for detection purposes. Preferably, thedetection group generates a fluorescent signal.

Molecular tags within a plurality are selected so that each has a uniqueseparation characteristic and/or a unique optical property with respectto the other members of the same plurality. In one embodiment, thechromatographic or electrophoretic separation characteristic isretention time under a set of standard separation conditionsconventional in the art, e.g., voltage, column pressure, column type,mobile phase or electrophoretic separation medium. In anotherembodiment, the optical property is a fluorescence property, such asemission spectrum, fluorescence lifetime or fluorescence intensity at agiven wavelength or band of wavelengths. Preferably, the fluorescenceproperty is fluorescence intensity. One or two or more of the moleculartags of a plurality may have identical migration or retention times, butthey will have unique fluorescent properties, e.g. spectrally resolvableemission spectra, so that all the members of the plurality aredistinguishable by the combination of molecular separation andfluorescence measurement.

Preferably, released molecular tags are detected by electrophoreticseparation and the fluorescence of a detection group. In suchembodiments, molecular tags having substantially identical fluorescenceproperties have different electrophoretic mobilities so that distinctpeaks in an electropherogram are formed under separation conditions.Preferably, pluralities of molecular tags of the invention are separatedby a conventional capillary electrophoresis apparatus, either in thepresence or absence of a conventional sieving matrix. During or afterelectrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absenceand/or amounts of molecular tags are measured by using one or morestandards.

A cleavage-inducing moiety, or cleaving agent, is a group that producesan active species that is capable of cleaving a cleavable linkage,preferably by oxidation. Preferably, the active species is a chemicalspecies that exhibits short-lived activity so that its cleavage-inducingeffects are only in the proximity of the site of its generation. Eitherthe active species is inherently short lived, so that it will not createsignificant background beyond the proximity of its creation, or ascavenger is employed that efficiently scavenges the active species, sothat it is not available to react with cleavable linkages beyond a shortdistance from the site of its generation. Illustrative active speciesinclude singlet oxygen, hydrogen peroxide, NADH and hydroxyl radicals,phenoxy radical, superoxide and the like. Illustrative quenchers foractive species that cause oxidation include polyenes, carotenoids,vitamin E, vitamin C, amino acid-pyrrole N-conjugates of tyrosine,histidine and glutathione. See, e.g. Beutner et al., 2000, Meth.Enzymol. 319:226-241.

One consideration in designing assays employing a cleavage-inducingmoiety and a cleavable linkage is that they not be so far removed fromone another when bound to a receptor complex that the active speciesgenerated by the cleavage-inducing moiety cannot efficiently cleave thecleavable linkage. In one embodiment, cleavable linkages preferably arewithin about 1000 nm and preferably within about 20-200 nm of a boundcleavage-inducing moiety. More preferably, for photosensitizercleavage-inducing moieties generating singlet oxygen, cleavable linkagesare within about 20-100 nm of a photosensitizer in a receptor complex.One of ordinary skill in the art will recognize that the effectiveproximity of a particular sensitizer may depend on the details of aparticular assay design and may be determined or modified by routineexperimentation.

A sensitizer is a compound that can be induced to generate a reactiveintermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed by Di Mascio et al., 1994, FEBS Lett. 355:287 and Kanofsky,1983, J. Biol. Chem. 258:5991-5993; Pierlot et al., 2000, Meth. Enzymol.319:3-20.

Photosensitizers may be attached directly or indirectly, via covalent ornon-covalent linkages, to the antibodies. Guidance for constructing suchcompositions are available in the literature, e.g. in the fields ofphotodynamic therapy, immunodiagnostics and the like. Exemplary guidancemay be found in Ullman et al., 1994, Proc. Natl. Acad. Sci. USA 91,5426-5430; Strong et al., 1994, Ann. New York Acad. Sci. 745: 297-320;Yarmush et al., 1993, Crit. Rev. Therapeutic Drug Carrier Syst. 10:197-252; and U.S. Pat. Nos. 5,709,994, 5,340,716, 6,251,581, and5,516,636.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonochromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation depends on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation and its distance from the sample. In general, theperiod for irradiation may be less than about a microsecond to as longas about 10 minutes, usually in the range of about one millisecond toabout 60 seconds. The intensity and length of irradiation should besufficient to excite at least about 0.1% of the photosensitizermolecules, usually at least about 30% of the photosensitizer moleculesand preferably, substantially all of the photosensitizer molecules.Exemplary light sources include lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps and incandescentlamps such as, e.g., tungsten and tungsten/halogen and flashlamps. Anexemplary photoactivation device suitable for use in the methods of theinvention is disclosed International Patent Publication No. WO03/051669, which is incorporated by reference herein, including anydrawings. In such embodiments, the photoactivation device is an array oflight emitting diodes (LEDs) mounted in housing that permits thesimultaneous illumination of all the wells in a 96-well plate.

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and those disclosedby U.S. Pat. Nos. 5,536,834, 5,763,602, 5,565,552, 5,709,994, 5,340,716,5,516,636, 6,251,581 and 6,001,673; published European PatentApplication No. 0484027; Martin et al., 1990, Methods Enzymol.186:635-645 and Yarmush et al., 1993, Crit. Rev. Therapeutic DrugCarrier Syst. 10:197-252, all of which are incorporated by referenceherein, including any drawings. As with sensitizers, in certainembodiments, a photosensitizer may be associated with a solid phasesupport by being covalently or non-covalently attached to the surface ofthe support or incorporated into the body of the support. In general,the photosensitizer is associated with the support in an amountnecessary to achieve the necessary amount of singlet oxygen. Generally,the amount of photosensitizer is determined empirically according toroutine methods.

Following cleavage, the sample can then be analyzed to determine theidentity of molecular tags that have been released. Where an assayemploying a plurality of binding compounds is employed, separation ofthe molecular tags will generally precede their detection. The methodsfor both separation and detection are determined in the process ofdesigning the molecular tags for the assay. A preferred mode ofseparation employs electrophoresis, in which the various tags areseparated based on known differences in their electrophoreticmobilities.

In a fourth aspect, the invention is drawn to a purified antibody thatbinds to p95. In a preferred embodiment, the purified antibody bindsspecifically to p95. In a preferred embodiment, the antibody bindsspecifically to the extracellular domain of p95 but not full lengthHER2. In a preferred embodiment, the antibody is a polyclonal antibodyor a monoclonal antibody. In a preferred embodiment, the antibody is amonoclonal antibody. In a preferred embodiment, the antibody was raisedagainst one of the peptides having SEQ ID NOs 1-7. In a preferredembodiment, the invention is drawn to one of the peptides having SEQ IDNOs 1-7. In certain embodiments, the antibody is or comprises one of theantibodies produced by hybridoma cell lines deposited with the ATCChaving accession number PTA-9738 (p95.D3.4), PTA-9739 (p95.D8.2) andPTA-9740 (p95.D9.1). In one embodiment, the antibody is p95.D9.1.

In a preferred embodiment, the invention is drawn to the DNA encodingthe antibody or peptides. The DNA encoding the monoclonal antibodies isisolated and sequenced using techniques commonly known to those skilledin the art of cloning. Once isolated, the DNA can be ligated intoexpression vectors and transfected into appropriate host cells to obtainrecombinant antibodies from cultured cells (see Plueckthun (1992)Immunological Rev. 130: 151-188).

Those with skill in the art will appreciate that the amino acid sequenceof an antibody or peptide can be modified and that modifications may bedesirable to enhance the properties of the antibody for therapeutic,analytical or diagnostic use. Further it will be appreciated that one ormore amino acids in these antibodies or antibodies may be changed byinsertion, deletion or substitution without appreciably diminishing thebinding characteristics of the antibody. Exemplary amino acid changeswould be substitutions using amino acids with similar molecularcharacteristics (i.e., conservative substitutions, e.g., changing aminoacids from within the following subgroups of aromatic amino acids,acidic amino acids, basic amino acids or amino acids with amides orsulphurs). Other non-conservative substitutions or insertions may bemade without appreciably altering molecular integrity or bindingcharacteristics. Further, some amino acid changes or collection of aminoacid changes will enhance properties of the antibody, including but notlimited to, better binding affinity, greater stability, (e.g.,resistance to proteases) and/or ease of production. Methods for changingamino acid sequences and/or selecting for molecules with betterproperties are known to those with skill in the art. Preferably, inintact antibodies or peptides, the degree of sequence identity aftermodification is at least 50% and more preferably, at least 75% and mostpreferably at least 90-95%. Each of these antibodies or peptides isintended to be within the scope of the contemplated invention.

In a preferred embodiment, antibodies targeted to p95 or peptides may beused to develop additional p95-targeted molecules. Modifications of theantibodies described herein may be desirable to improve qualitiesincluding, but not limited to, increasing function, decreasingimmunogenicity, increasing stability, improving pharmacologic propertiessuch as serum half-life and aiding in ease and yield of production. Eachof these targeted molecules is intended to be within the scope of thecontemplated invention.

In a preferred embodiment, humanized antibodies comprising the antigenbinding regions of the antibodies described herein (ATCC # PTA-9738,PTA-9739 and PTA-9740) in a human framework may be used for therapeuticapplications. Several methods for humanizing antibodies have beenreported (see Jones et al. (1986) Nature 321:522-525, Riechmann et al.(1988) Nature 332:323-327, Verhoeyen et al. (1988) Science239:1534-1536). Typically, the non-human sequences of the variabledomain are screened computationally against the entire repertoire ofhuman light and heavy chain variable domain sequences to find the humanvariable framework sequences closest to the rodent sequences (see Simset al. (1993) J. Immunol. 151:2296-2308, Chothia et al. (1987) J. Mol.Biol. 186:901-917). Alternatively, consensus frameworks can be used (seeCarter et al. (1992) Proc. Natl. Acad. Sci. USA 89:4285-4289 and Prestaet al. (1993) J. Immunol. 151:2623-2632). In a preferred embodiment,computer-aided design is used to select sequences that confer stabilityand retain or improve binding characteristics. Each of these is intendedto be within the scope of the contemplated invention.

In another embodiment, the antibody complementarity determining regions(CDRs) may be used to create targeted binding molecules that bind thesame epitope in p95 but are contained within a framework that is not anative antibody. For example, one skilled in the art would appreciatethat methods are available for creating binding molecules in which theframework may be a portion of an antibody, for example, an scFv orF(ab′)₂ (see WO 93/16185 and Carter et al. (1992) Bio/Technology10:163-167, respectively), each of which is incorporated by referenceherein. One skilled in the art may also appreciate that a completelyunrelated protein (such as a bacterial beta-lactamase) can properlydisplay the binding domain(s) to form a binding compound. In this sense,related antibodies, as defined herein, are intended to be within thescope of the invention.

The antibody may act therapeutically through binding alone or throughother properties (e.g., enzymatic activity or toxic warheads). In oneembodiment, the targeted protein may be modified to exert a therapeuticeffect or a greater therapeutic effect via antigen-dependentcell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity(CDC). In another embodiment, toxins may be conjugated to the antibodyor targeted protein. Exemplary small molecule toxins include but are notlimited to maytansine, calicheamicin and CC-1065 (see, e.g., Carter andSenter (2008) Cancer J. 14:154-169). Additionally, radiolabels can belinked to antibodies to create targeted therapeutics. Biologic toxinsmay also be linked to targeted proteins and include, but not be limitedto, diphtheria toxin, Pseudomonas exotoxin, abrin and ricin (seeKreitman (2006) AAPS J. 18:E532-551).

In a further embodiment, the targeted antibodies (or fragments thereof)may be fused to enzymes for use in antibody-directed enzyme prodrugtherapy (ADEPT; see Bagashawe (1987) Br. J. Cancer 58:700-703 and Senteret al. (1988) Proc. Natl. Acad. Sci. USA 85:4842-4846). In anotherembodiment, the antibodies or targeted proteins may be fused tomolecules such as polyethylene glycol, that enhance pharmacologicproperties, such as serum half-life (see Harris and Chess (2003) Nat.Rev. Drug Discov. 2:214-221).

In a fifth aspect, the invention is drawn to a method for determiningwhether a subject with a cancer is likely to respond to treatment with atargeted therapy, for predicting a time course of disease and/or forpredicting probability of a significant event in the time course of thesubject's cancer based on a measurement of an amount of p95 or a p95complex in a sample. In one embodiment, the invention is drawn to amethod for determining whether a subject with a cancer is likely torespond to treatment with a Her-2 acting agent. In another embodiment,the method is drawn to a method of predicting a time course of a diseasein a subject with a cancer. In another embodiment, the method is drawnto predicting the probability of a significant event in a subject with acancer.

In a preferred embodiment, a time course is measured by determining thetime between significant events in the course of a patient's disease,wherein the measurement is predictive of whether a patient has a longtime course. In a preferred embodiment, the significant event is theprogression from primary diagnosis to death. In a preferred embodiment,the significant event is the progression from primary diagnosis tometastatic disease. In a preferred embodiment, the significant event isthe progression from primary diagnosis to relapse. In a preferredembodiment, the significant event is the progression from surgery todeath. In a preferred embodiment, the significant event is theprogression from surgery to relapse. In a preferred embodiment, thesignificant event is from surgery to metastases. In a preferredembodiment, the significant event is the progression from metastaticdisease to death. In a preferred embodiment, the significant event isthe progression from metastatic disease to relapse. In a preferredembodiment, the significant event is the progression from relapse todeath. In a preferred embodiment, the time course is measured withrespect to overall survival rate, time to progression and/or using theRECIST or other response criteria.

Her2-positive tumors are not all responsive to trastuzumab and othertherapeutics that bind to epitopes in the extracellular domain ofmembrane-bound Her2. An explanation for a lack of responsiveness may bethat cleavage of the extracellular domain removes the binding site fortrastuzumab and like therapeutics, and leaves p95 with a constitutivelyactive tyrosine kinase activity. High levels of Her2 and relatively highlevels of p95, therefore, may be a meaningful marker for the likelihoodthat a tumor will fail to respond to trastuzumab and other therapeuticsthat bind to epitopes on the extracellular domain of membrane-boundHer2.

In certain embodiments, the method comprises measuring in a biologicalsample from the subject's cancer an amount of p95, wherein if the amountof p95 is low, then the patient is likely to respond to the Her-2 actingagent and/or the patient has a long time course. In certain embodiments,the biological sample comprises FFPEs. In certain embodiments, thesubject's cancer is breast cancer. In certain embodiments, the breastcancer is metastatic. In certain embodiments, the Her-2-acting agent istrastuzumab. In certain embodiments, the assay is the VeraTag assay. Incertain embodiments, likeliness to respond is measured with respect tooverall survival rate, time to progression and/or using the RECISTcriteria.

In certain embodiments, a predetermined measure is created by dividingpatient cohorts into at least two patient subgroups. In certainembodiments, the number of subgroups is two so that the patient sampleis divided into a subgroup of patients whose p95 is high and a subgroupwhose p95 is low; the amount of p95 in the subject is compared to eitherthe high subgroup or the low subgroup; if the amount of p95 in thepatient is high, then the patient is not likely to respond to a Her-2acting agent that is not a p95-acting agent and/or the patient is likelyto have a short time course; if the amount of p95 is low (and the amountof Her2 is high), then the patient is likely to respond to Her-2 actingagents and the time course may be long. In certain embodiments, thenumber of subgroups is greater than two, including, without limitation,three subgroups, four subgroups, five subgroups and six subgroups. Incertain embodiments, likeliness to respond or time course is measuredwith respect to overall survival rate, time to progression and/or usingthe RECIST criteria. In certain preferred embodiments, the Her-2 actingagent that is not a p95-acting agent is trastuzumab.

In certain embodiments, the predetermined measure is an optimal cutoff.In certain embodiments, the amount of p95 in the subject is compared tothe optimal cutoff; if the amount of p95 in the patient is high, thenthe patient is not likely to respond to a Her-2 acting agent that is nota p95-acting agent and/or the patient's time course is likely to beshort. Any method known to one of skill in the art to be useful fordetermining an amount of p95 expression can be used in accordance withthe present invention. Such methods may include any method disclosedherein such as, for example, without limitation, VeraTag, FRET, BRET,Biomolecular Fluoresence Complementation, Proximity Ligation Assay andRolling Circle Amplification.

In a preferred embodiment, the subject's cancer is breast cancer. In apreferred embodiment, the Her-2 acting agent is a tyrosine kinaseinhibitor and if the amount of p95 is high, then the patient is likelyto respond to the targeted therapy, the patient is likely to have a longtime course and/or the patient is not likely to have a significantevent. In a preferred embodiment, the targeted therapy is an inhibitor,such as a protease inhibitor, and if the amount of p95 is high, then thepatient is likely to respond to the targeted therapy, the patient islikely to have a long time course and/or the patient is not likely tohave a significant event.

In certain embodiments the Her2-acting agent is selected from the groupconsisting of pertuzumab, trastuzumab, canertinib, lapatinib,mubritinib, AEE-788, HKI-272, BIBW-2992, and BMS-599626. See e.g.,Spector, 2007, Breast Cancer Res. 9:205. In a preferred embodiment, theHer-2-acting agent is trastuzumab (Herceptin®). See, e.g., Goldenberg,1999, Clin Ther. 21:309-18; and Shak, 1999, Semin Oncol. 26:71-7.

In a preferred embodiment, the inhibitor inhibits metalloproteasesincluding, but not limited to, matrix metalloproteases and/or member(s)of the ADAM family of proteases. In a preferred embodiment, theinhibitor inhibits ADAM10. Members of the ADAM family ofmetalloproteases are thought to mediate cleavage of erbB family members;specifically, ADAM10 (see Gee et al. (2003) Breast Cancer Res. 5:223-224and Sahin (2004) J. Cell Biol. 164:769-779) is thought to be a majorsource of Her2 ECD sheddase activity (see Liu, P C et. al (2006) CancerBiology and Therapy 6: 657-664) and is a target for therapeuticintervention.

In certain embodiments, the subject may be administered a combinationtherapy that includes trastuzumab. The combination therapy can includetrastuzumab in combination with one or more of any chemotherapeuticagent known to one of skill in the art without limitation (see, forexample, Romond, E H, N Engl J Med (2005) 353(16):1673, which isincorporated by reference herein, including any drawings). Preferably,the chemotherapeutic agent has a different mechanism of action fromtrastuzumab. Particular examples of chemotherapeutic agents that can beused in the various embodiments of the invention, includingpharmaceutical compositions, dosage forms, and kits of the invention,include, without limitation, cytarabine, melphalan, topotecan,fludarabine, etoposide, idarubicin, daunorubicin, mitoxantrone,cisplatin, paclitaxel and cyclophosphamide.

In another embodiment, the invention provides an analytical method forscreening therapeutic candidates for potential efficacy as therapeuticagents. In a preferred embodiment, the VeraTag assay, as describedherein, can be employed to test for p95 levels in biological systems (orderivatives thereof, such as cell lines) that have been treated withputative inhibitors of enzymes (e.g., sheddases) that are thought to beinvolved in generating p95-dependent growth of tumors. Candidates withinhibitory activity will show decreased levels of p95 in the VeraTagassay as described herein. In a preferred embodiment, the enzymestargeted by the putative inhibitors are matrix metalloproteases. In apreferred embodiment, the enzymes are members of the ADAM family ofproteases. In a preferred embodiment, the enzyme is ADAM10.

In a further aspect, the invention provides methods of treating asubject with cancer. In one aspect, the methods comprise determiningthat the subject is afflicted with a cancer that is likely to respond totreatment and/or has a long time course according to a method of theinvention, and administering an effective amount of compound to thesubject as a result of said determination. In another aspect, themethods comprise determining that a subject is afflicted with a cancerthat is likely to respond to treatment according to a method of theinvention, then advising a medical professional of the treatment optionof administering to the subject an effective amount of an agent. Inanother embodiment, the agent is at least two agents.

EXAMPLES Example 1 Generation of Expression Vectors for Tagged p95 andHER2

Expression vectors for p95 (pcDNA6myc/hisA M611-p95) and full-lengthHER2 (pcDNA6-HER2) were constructed using the pcDNA6A-myc/his vectorfrom Invitrogen. The HER2 expression sequence included a hemagglutinin(HA) tag (expressed amino acids: YPYDVPDYA; SEQ ID NO:8) two amino acidsdownstream of the putative leader sequence cut site. A stop codon wasincluded at the end of the HER2 sequence to prevent the incorporation ofthe myc/his tags embedded in pcDNA6A-myc/his. The p95 sequence startedfrom Methionine-611, numbered from the HER2 amino acid sequence.Upstream from Methionine-611 was placed sequence encoding the HER2leader sequence plus two amino acids followed by the same 9-amino acidHA tag used in the HER2 expression vector. A stop codon was not includedat the end of the p95 sequence so that the myc/his tags would beincluded in the expressed protein.

Example 2 Generation of antibodies against p95

The following monoclonal antibodies/hybridomas of the present inventionare described below:

P95.A3, p95.B1, p95.B2, p95.B3, p95.B4, p95.B5, p95.B6, p95.B7, p95.B8,p95.B9, p95.B10, p95.B11, p95.B12, p95.B13, p95.B14, p95.B15, p95.B16,p95.B19, p95.B20, p95.B21, p95.B23, p95.B24, p95.B25, p95.B26, p95.D2,p95.D3, p95.D4, p95.D5, p95.D6, p95.D7, p95.D8, p95.D9, p95.D10,p95.D11, p95.D12, p95.D13, p95.D102, p95.D111, p95.D112, p95.D115,p95.D117, p95.D119, p95.D120, p95.D121, p95.D123, p95.D125, p95.D126,p95.D127, p95.D128, p95.D129, p95.D131, p95.D132, p95.D133, p95.D134,p95.D135, p95.D137, p95.D139, p95.E1, p95.E2, p95.E3, p95.E4.If the monoclonal antibody has been cloned, it will get the nomenclature“X.1,” e.g., the first clone of p95.D9 will be referred to as D9.1, thesecond clone of D9 will be referred to as D9.2, etc. For the purposes ofthis invention, a reference to p95.D9 or D9 will include all clones,e.g., D9.1, D9.2, etc.

Mice were immunized with peptides representative of epitopes that arelikely to be present in p95. Peptides used for immunizations were:

P95.A peptide: (SEQ ID NO: 2) ASPLTSIIS P95.B peptide: (SEQ ID NO: 3)PAEQRASPLTSIIS P95.D peptide: (SEQ ID NO: 5) MPIWKFPDEEGA P95.E peptide:(SEQ ID NO: 6) PSGVKPDLSYMPIWKPeptides were conjugated to keyhole limpet hemocyanin (KLH) forimmunizations and bovine serum albumin (BSA) for screening using SMCCchemistry (Pierce, Rockford, Ill.).

Mice (Balb/c, FVB, C3H, or CD-1) were immunized with peptide-KLHconjugates twice weekly for 5 weeks to generate anti-p95 MAbs capable ofbinding to p95 in bodily fluids and on a cell surface. Immunizationswere done intradermally in both rear footpads with 10 μg peptide-KLHconjugate. Peptide-KLH conjugates were mixed with suitable adjuvantsprior to injection. Titermax (Sigma, St. Louis. Mo.) was used for thefirst injection; Adju-Phos (Accurate Chemical & Scientific Corp.,Westbury, N.Y.) was used for injections 2 to 9. For the 10^(th)injection, antigen was mixed with phosphate buffer saline.

Four days after the final immunization, lymphocytes were isolated frompopliteal lymph nodes and immortalized by electrofusion (electrofusiongenerator ECM2001; Harvard Apparatus, Holliston, Me.) with thecontinuous myeloma cell line P3x63Ag8.653 (Kearney, J F et al. (1979) JImmunology 123, 1548-1550). Fused cells were selected by culturing inselection medium (DMEM/15% FBS) containing 2.85 μM Azaserine, 50 μMHypoxanthine (HA) (Sigma) or 50 μM Hypoxanthine, 0.2 μM Aminopterin, 8μM Thymidine (HAT) (Sigma) supplemented with recombinant human IL-6(Sigma) at 0.5 ng/mL. Cultures were transitioned into medium (DMEM/10%FBS) without selection and IL-6 supplements for continued expansion andantibody production.

Hybridoma supernatants were screened by enzyme-linked solid phaseimmunoassay (ELISA), flow cytometry and western blotting for reactivityagainst p95. Monoclonal cultures were established after the screeningprocedure by single cell sorting using a flow cytometer.

Hybridoma supernatants were screened by direct ELISA using 100 ngpeptide-BSA conjugate or Her2-Fc fusion protein (R&D Systems,Minneapolis, Minn.) per well as antigen (Table 1). Her2-Fc is arecombinant protein containing the extracellular domain of Her2 fused tothe Fc domain of human IgG. Antibodies A3, B1, B2, B3, B4, B10, B11,B12, B13, B14, B15, B20, B21, B23, E1, E2, E3 and E4 reacted withpeptides p95.A and p95.B, and did not bind to Her2-Fc. Antibodies B5,B6, B8, B9, B16, B24, B25 and B26 reacted with peptide p95.B, but didnot bind to peptide p95.A and Her2-Fc. Antibodies B7 and B19 bound toboth peptides p95.A and p95.B, but binding to p95.A was much weaker thanbinding to p95.B. Antibodies D3, D5, D7, D10, D11, D13, D102, D120,D125, D126, D129, D131, D132, D133, D134 and D139 bound to peptidep95.D, and did not bind to Her2-Fc. Antibodies D2, D8, D9, D111, D115,D117, D119, D121, D123, D127, D128, D135 and D137 strongly bound top95.D peptide, and weakly bound to Her2-Fc. Antibodies D4, D6 and D12strongly bound to both p95.D peptide and Her2-Fc.

Table 1 shows the screening of conditioned media from the hybridomas bydirect ELISA. Wells were coated with 100 ng antigen (peptides p95.A,p95.B, p95.D, a negative control peptide unrelated in sequence to Her2,or Her2-Fc recombinant protein) and probed with hybridoma supernatants.Her2-Fc protein, containing the extracellular domain (ECD) of Her2, wasobtained from R&D Systems as a chimeric protein fused to the Fc regionof human IgG1. Bound antibody was detected with an alkalinephosphatase-conjugated goat-anti-mouse IgG antiserum. OD values forhybridoma supernatant/antigen pair are listed.

TABLE 1 Her2 Hybridoma p95.A p95.B p95.D Fc control supernatant peptidepeptide peptide protein peptide A3  1.393 2.027 0.131 0.118 B1  2.7533.495 0.096 0.119 B2  1.912 2.053 0.089 0.122 B3  0.913 0.928 0.0850.132 B4  1.835 2.482 0.103 0.143 B5  0.121 0.667 0.108 0.132 B6  0.1152.915 0.109 0.13 B7  0.358 3.251 0.107 0.135 B8  0.11 0.995 0.108 0.134B9  0.091 3.777 0.092 0.116 B10  2.212 3.03 0.095 0.115 B11  1.553 1.9160.092 0.118 B12  2.491 2.941 0.104 0.119 B13  1.495 1.679 0.122 0.144B14  3.693 3.858 0.129 0.14 B15  0.29 0.345 0.102 0.129 B16  0.108 1.780.097 0.124 B19  0.238 0.693 0.092 0.103 B20  0.447 0.855 0.102 0.114B21  0.515 0.7 0.109 0.124 B23  2.95 3.625 0.1 0.118 B24  0.112 3.4470.1 0.117 B25  0.089 1.556 0.093 0.104 B26  0.088 2.791 0.094 0.102 E1 3.7206 3.8345 0.1101 0.1102 E2  1.3195 1.8344 0.1023 0.0983 E3  2.72012.4782 0.1115 0.1106 E4  1.9273 1.9121 0.1168 0.1180 D2  1.4276 0.25550.0762 D3  2.3055 0.0890 0.1336 D4  3.9405 1.6825 0.1070 D5  3.41710.0939 0.1080 D6  0.9020 1.7200 0.0861 D7  2.0873 0.1152 0.0932 D8 3.7010 0.2191 0.0835 D9  3.4600 0.3109 0.0837 D10  3.1198 0.0865 0.0944D11  3.3918 0.1026 0.0814 D12  3.8707 4.0000 0.0921 D13  1.1890 0.09230.0956 D102 3.6168 0.0968 0.0925 D111 4.0000 0.6007 0.1107 D112 0.32470.4424 0.1043 D115 3.9346 0.4967 0.0932 D117 4.0000 0.5348 0.0930 D1194.0000 0.5448 0.1078 D120 0.6196 0.1175 0.1138 D121 3.7442 0.3166 0.1095D123 3.7323 0.3121 0.0899 D125 3.8027 0.0943 0.0934 D126 3.6641 0.09200.0913 D127 4.0000 0.4784 0.0858 D128 4.0000 0.4525 0.0984 D129 4.00000.1230 0.1074 D131 0.4459 0.1210 0.1077 D132 2.6356 0.1155 0.1059 D1333.9921 0.0954 0.0810 D134 3.7527 0.0934 0.1237 D135 2.5440 0.2019 0.0841D137 4.0000 0.3294 0.1063 D139 3.5412 0.1102 0.1096

A number of clones showed reactivity to the peptide and little to noreactivity toward HER2-ECD (D3, D5, D7-11 and D13 of FIG. 1). Othersshowed reactivity to both HER2-ECD and the immunization peptide (D4, D6and D12 of FIG. 1).

Conditioned media from clones were also used to stain blots ofpolyacrylamide gels run with lysates of SKBR3, 293T and 293T transfectedwith pcDNA6myc/hisA M611-p95 (see FIG. 2). 5 μg cell lysate from theSKBR3 and 293T cells or 1 μg cell lysate from the cells transfected withpcDNA6myc/hisA M611-p95 expression vector were separated on 4-12% NuPAGEgels (Invitrogen). The gels were blotted to PVDF membranes that werestained with conditioned media from hybridomas D4, D8, D12 or Her2 Ab8.Her2 Ab8 (Labvision, Fremont, Calif.) was used as positive controlantibody and binds to an intracellular epitope of Her2 that is also partof p95. Bound antibodies were detected with a horseradishperoxidase-conjugated anti-mouse IgG antiserum and an ECL reagent. InWestern blots, only D4, D8 and D12 showed significant binding,recognizing both full-length Her2 (in SKBR3 cell lysates) and p95 (inlysates of 293T cell transfected with p95 expression vectors).Antibodies A3, D3, D5, D6, D7, D9, D10, D11, D13 and all B and Eantibodies did not produce specific signals in western blots (data notshown). Anti-HER2 Ab8 from Labvision was included as a positive control(see FIG. 2).

Hybridoma supernatants were screened by flow cytometry using HEK293Fcells transiently transfected with HA-tagged, full-length Her2 orHA-tagged PcDNA6-p95. 293F cells were transfected using 293-fectin(Invitrogen, Carlsbad, Calif.) and incubated for 2 days. Cells wereeither directly used for staining or were fixed with paraformaldehydebefore staining Hybridoma supernatants were added to cells. Boundantibodies were detected using a biotinylated anti-mouse IgG serum andstreptavidin-phycoerythrin (native cells; Table 2, FIG. 3 a) or afluorescein-conjugated anti-mouse IgG serum (fixed cells; Table 3, FIG.3 b).

Antibodies could be roughly grouped into two classes based on binding top95 or HER2 expressing 293 cells. Antibodies A3, D3, D5, D6, D7, D10 andD11 bound to native cells expressing pcDNA6-p95, but did not bind tocells expressing full-length Her2. Antibodies D4, D8, D9 and D12 boundto native cells expressing pcDNA6-p95 and to native cells expressingfull-length Her2. Although full-length Her2 was recognized by theseantibodies, binding was relatively weak as compared to the positivecontrol antibodies HA.A28.2.

Table 2 shows the screening of hybridoma supernatants by flow cytometrywith native cells. 293F cells transfected with HA-Her2 or pcDNA6-p95 orcontrol 293F cells were stained with hybridoma supernatants. Meanfluorescence intensities (MFI), the percentages of stained cells and theMFI ratios are listed. The MFI ratio is the ratio of the MFI of aspecific hybridoma supernatant and the MFI of the negative anti-ricincontrol antibody. Antibody HA.A28.2 specific for the HA-tag was used aspositive control.

TABLE 2 293F HA-Her2 293F pcDNA6-095 Control 293F % % % stained MFIstained MFI stained MFI samples MFI cells ratio MFI cells ratio MFIcells ratio anti-ricin 0.29 0.8% 1.0 0.24 0.9% 1.0 0.26 0.9% 1.0HA.A28.2 19.98 94.3% 68.9 15.79 74.4% 65.8 0.40 17.7% 1.5 A3 0.39 1.7%1.3 6.83 53.5% 28.5 0.31 1.6% 1.2 D3 0.36 5.8% 1.2 24.15 83.6% 100.60.27 1.3% 1.0 D4 2.80 80.0% 9.7 26.88 88.5% 112.0 0.33 11.0% 1.3 D5 0.260.5% 0.9 1.19 35.3% 5.0 0.27 2.5% 1.0 D6 0.32 2.6% 1.1 0.50 26.6% 2.10.26 1.7% 1.0 D7 0.44 10.7% 1.5 7.90 65.6% 32.9 0.34 15.1% 1.3 D8 3.1661.9% 10.9 26.58 81.3% 110.8 0.28 3.6% 1.1 D9 3.37 48.1% 11.6 22.8976.8% 95.4 0.28 3.2% 1.1 D10 0.30 3.2% 1.0 16.28 73.7% 67.8 0.25 1.2%1.0 D11 0.40 5.7% 1.4 7.22 60.5% 30.1 0.27 2.2% 1.0 D12 3.17 83.6% 10.924.17 85.3% 100.7 0.45 35.4% 1.7 D13 0.26 0.2% 0.9 0.24 0.6% 1.0 0.271.6% 1.0

Antibodies A3, D5, D6, D7, D11 and D13 did not bind to formalin-fixed293F cells expressing pcDNA6-p95 indicating that the fixation proceduremodified the epitopes recognized by these antibodies. Antibodies D4 andD12 bound well to fixed 293F cells expressing either pcDNA6-p95 or Her2.Antibodies D3, D8, D9 and D10 bound well to fixed 293F cells expressingpcDNA6-p95, but did not bind or bound weakly to fixed cells expressingHer2.

Table 3 shows the screening of hybridoma supernatants by flow cytometrywith formalin-fixed cells. 293F cells transfected with HA-Her2 orpcDNA6-p95 or control 293F cells were formalin-fixed and stained withhybridoma supernatants. Mean fluorescence intensities (MFI), the percentof stained cells and the MFI ratio are listed. The MFI ratio is theratio of the MFI of a specific hybridoma supernatant and the MFI of thenegative anti-ricin control antibody. Antibody HA.A28.2 specific for theHA-tag was used as positive control.

TABLE 3 293F HA-Her2 293F pcDNA5-p95 Control 293F % stained MFI %stained MFI % stained MFI samples MFI cells ratio MFI cells ratio MFIcells ratio anti-ricin 0.48 1.0% 1.0 0.48 0.9% 1.0 0.43 0.9% 1.0HA.A28.2 2.12 60.8% 4.4 1.96 36.6% 4.1 0.56 12.3% 1.3 D3 0.54 4.2% 1.11.49 27.4% 3.1 0.47 3.2% 1.1 D4 1.21 35.2% 2.5 2.24 38.4% 4.7 0.49 6.3%1.1 D5 0.33 0.3% 0.7 0.36 1.8% 0.8 0.29 0.2% 0.7 D6 0.33 0.1% 0.7 0.330.2% 0.7 0.29 0.1% 0.7 D7 0.42 0.6% 0.9 0.50 6.6% 1.0 0.40 0.8% 0.9 D80.79 21.0% 1.6 2.07 32.6% 4.3 0.43 1.8% 1.0 D9 0.71 18.4% 1.5 2.08 32.1%4.3 0.34 0.3% 0.8 D10 0.48 1.7% 1.0 1.47 29.7% 3.1 0.31 0.1% 0.7 D110.42 1.9% 0.9 0.55 12.6% 1.1 0.36 0.3% 0.8 D12 0.99 27.1% 2.1 2.00 32.3%4.2 0.33 0.2% 0.8 D13 0.43 2.4% 0.9 0.58 0.9% 1.2 0.30 0.1% 0.7anti-ricin 0.48 1.0% 1.0 0.48 0.9% 1.0 0.43 0.9% 1.0

Example 3 Production of FFPE Slides from Cell Lines with and withoutExpression of p95 Transgene

Three breast cancer cell lines, MCF-7, MDA-MB-453 and SKBR-3, werepurchased from American Type Cell Culture Collection. MCF-7-p95c (where“c” indicates a clonal line), MCF7-HER2c and SKBR3-p95c were obtainedfrom the laboratory of Jose Baselga. These clones were made from aslightly different form of p95 containing no leader sequence or HA tag,as described in Anido et al. (2006) EMBO J., 25:13, 3234. Thep95-containing cell lines were generated by the transfection of parentalMCF7 or SKBR3 cells with an expression vector containing a HindIIfragment of full-length HER2 that allows translational initiation fromseveral internal methionines, including Met611. MCF-7, MCF7-p95c,MCF7-HER2c and SKBR3-p95c were maintained at 37° C. in 5% CO₂ in 50:50Dulbecco's modified Eagle medium (DMEM): F12, 10% fetal bovine serum(FBS), 1% penicillin-streptomycin (PS), 10 μg/mL bovine insulin and 2 mML-glutamine. MDA-MB-453 and SKBR-3 were maintained at 37° C. in 5% CO₂in 50:50 DMEM: F12, 10% FBS, 1% PS and 2 mM L-glutamine. Cells wereplated at a density of 30 million per 500 cm². Cells intended fortransient transfection were allowed to attach for 4 hours. The cellswere transfected with Fugene HD (Roche) according to the manufacturer'sinstructions. The culture media was changed after 1 day and the cellswere fixed on day 2. After removal of medium, the cells were washed oncewith 50 mL cold D-PBS (Invitrogen) and fixed with 50 mL of 10% NBF(neutral buffered formalin). Cells were left in a 4° C. cold room for 30minutes then scraped from the culture plates. The cell slurry wascollected into 50 mL centrifuge tubes and pelleted at 3200×g for 15 min.The cell pellet was transferred to a rubber O-ring, wrapped with filterpaper and placed in a processing cassette. Automatic Tissue-Tekprocessor was used for processing. Briefly, the cell pellet was exposedto increasing concentrations of alcohol, Clear-rite (xylene substitute)and paraffin. After processing, pellet was embedded in a block using aparaffin embedding station. All solvents used for cell pellet processingwere obtained from Richard-Allen Scientific.

Sections of 5 um in thickness were sliced with a microtome (LEICA) andplaced on positively charged glass slides (VWR) with serial numberlabeled. Slides were air-dried for 30 min and then baked at 60° C. for 1hr. All sample slides were stored at 4° C. for future use.

To verify expression of transgenes in transfected cells made into FFPEblocks, cell lysates were prepared from samples of cells removed andlysed just prior to the addition of NBF described above. Lysis Buffercontained 1% Triton X-100, 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 50 mMNaF, 50 mM sodium beta-glycerophosphate, 1 mM Na₃VO₄, 5 mM EDTA, 10μg/mL pepstatin and 1 tablet Complete Protease Inhibitor (Roche#1836170) in 10 mL water. Samples were mixed with 2× Laemmli buffer(Biorad) and heated to 70° C. for 10 minutes. Separately, proteincontent was assessed by bicinchoninic acid (BCA) assay (Pierce)according to the manufacturer's instructions. Twenty micrograms ofprotein per lane were loaded into a 4-12% gradient gel and run in aMOPS-based running buffer (Invitrogen) at 180 v for approximately 1hour. Bands were transferred to a nitrocellulose membrane (Invitrogen)in Nu-PAGE transfer buffer (Invitrogen)+10% methanol at 45 v for 1.5hours. The membrane blots were first blocked with PBST (1% Triton-X100in PBS) plus 3% nonfat dry milk for 30 minutes with gentle shakingFollowing two 15 minute washes with PBST, blots were treated with 1.0μg/mL anti-HER2 Ab8 in PBST plus 0.03% nonfat dry milk overnight withgentle shaking Following two 15 minute washes with PBST, blots were nexttreated with a 1:50,000-fold dilution of a goat anti-mouse linked withhorseradish peroxidase (Pierce #31430) with gentle shaking for 30minutes. Following two 20 minute washes with PBST the blots weredeveloped with the West Dura HRP detection kit (Pierce) according to themanufacturer's instructions with images captured on film (Kodak). FIG. 4shows results from a representative set of cells that were subsequentlymade into FFPE blocks then cut into slides. Lane 1 shows an expectedsmall amount of material in the p95 region that is not found in the MCF7lane 5 (Anido et al. (2006) EMBO J. 25:13, 3234). Some evidence of thisweak p95 band is also seen in the transient-transfected MCF7-HER2 oflane 6 using a 50:50 weight mix of pcDNA6-HER2 and pcDNA6 vector. Lanes2-4 show the expected expression of full-length HER2 and p95 in theclonal lines. The cells assayed in Lane 7 were transfected with the sameamount of pcDNA6-HER2 as the cells in lane 6 but with pcDNA6myc/hisAM611-p95 substituted for the empty vector. Lane 8 shows the results ofMCF7 cells transfected with 100% pcDNA6myc/hisA M611-p95. Lane 9 showsSKBR3 cells, useful for comparison against lanes 3 and 4. Theuntransfected SKBR3 shows a band in the lower region of the p95 regionbut not the upper bands shown in the transfected lines. This is notunexpected as SKBR3 is known to shed some amount of HER2-ECD (Zabreckyet al. (1991) J. Biol. Chem. 266:3 1716).

Example 4 Screening of Anti-p95 Antibodies by Veratag Assay

The monoclonal antibody Ab8 against cytoplasmic domain of HER2 waspurchased from Lab Vision. A goat anti-mouse antibody was purchased fromThermo Scientific (#31164) VeraTag reporters (Pro125 and Pro14) weresynthesized and purified according to protocol described previously(See, for example, above and U.S. Pat. No. 7,105,308, which isincorporated by reference herein, including any drawings).Antibody-VeraTag and antibody-biotin conjugates, i.e., Ab8-biotin, goatanti-mouse-Pro125 anti-p95-Pro125, were made usingsulfo-NHS-LC-LC-biotin (Pierce) as linker according to manufacturer'sprotocol and conjugation products purified by HPLC (Agilent) or PD-10desalting column (GE).

A p95 FFPE assay was carried out essentially as shown in FIG. 5 a. FFPEsamples were deparaffinized/rehydrated using a series of solvents.Briefly, slides were sequentially soaked in xylene (2×, 5 min), 100%ethanol (2×, 5 min), 70% ethanol (2×, 5 min) and deionized water (2×, 5min). Heat-induced epitope retrieval of the rehydrated samples wasperformed in a dish containing 250 mL of 1× Diva buffer (Biocare Medical#DV2004MM) using microwave oven (Spacemaker II, GE): 3.5 min at power 10followed by 10 min at power 3. After being cooled down for 1 hour atroom temperature, the slides were rinsed six times with deionized water.Slides were partially dried in a centrifuge (Tomy PMC-082) modified tospin slowly. A hydrophobic circle was drawn on the slide using ahydrophobic pen (Zymed) to retain reagents on slides. The samples werethen blocked for 1 hr with Blocking Buffer that contained 10% goat serum(Sigma #S1000) and 1.5% bovine serum albumin in 1×PBS. After removal ofthe Blocking Buffer with aspiration, a solution containing the anti-p95antibody in Blocking Buffer was added to the slides and left at 4° C.overnight in a humidified chamber with gentle shaking. The concentrationof anti-p95 antibody was 1.0 μg/mL for screening of multiple antibodies,otherwise 4 μg/mL of D9.1 antibody was used. The antibody solution wasaspirated and samples were washed with PBS containing 0.25% TritonX-100for 5 minutes then PBS alone for 5 minutes. Following aspiration, 50 μLof 1.0 μg/mL goat anti-mouse antibody labeled with VeraTag in BlockingBuffer was added. This antibody was allowed to incubate at roomtemperature for 1.5 hours in a humidified chamber. The slides were nextrinsed with deionized water followed by PBS containing 0.25% TritonX-100for 5 minutes. Slides were then loaded onto racks and submerged indeionized water 6 times. Following centrifugation of the slides, 100 μLCapture Buffer containing 1.0 mM dithiothreitol (DTT), 3 pM fluoresceinand two CE internal markers (MF and ML) in 0.01×PBS was added on samplesections. Slides were incubated in a humidified chamber for 2 hours toallow for the release of the VeraTag. Capture Buffer from each slide wastransferred to a CE 96-well plate then diluted appropriately (generally10-fold) in Capture Buffer not containing DTT. The released VeraTagreporters in the CE samples were separated and detected on a ABI3100 CEinstrument (22-cm capillary array, Applied Biosystems) under the CEinjection condition of 6 kV and 50 sec and run for 650 seconds at 30° C.

The identification and quantification of VeraTag was carried out usingVeraTag Informer software (see, for example, United States publicationnumber 0203408-A1, which is incorporated by reference herein, includingany drawings). To analyze the VeraTag signals in a raw CEelectropherogram, two CE internal markers, MF (first marker) and ML(last marker), were used to identify the VeraTag peaks according totheir electrophoretic mobility or migration time, t, relative to the twomarkers, i.e., [t(VeraTag)-t(MF)]/[t(ML)-t(MF)]. The identified VeraTagpeaks were then quantified by peak area calculation for each VeraTag. Tocorrect for variability in VeraTag recovery from the tissue section, andthe run variability in CE injection efficiency and/or detectionsensitivity across capillary array, fluorescein (3 pM) was included inthe VeraTag Capture Buffer (CB), and co-electrophoresed as an internalreference control in each sample run. The area of each VeraTag peak wasthen reported as relative peak area (RPA) by area normalization of theVeraTag peak (VeraTag peak area) to the internal fluorescein peak(fluorescein peak area).

Slides were stained with hematoxylin and eosin (H&E) by standardtechniques. Briefly, slides were placed in staining racks and firstrinsed in tap water. Slides were serially dipped in hematoxylin,clarifier and bluing agent for 45 seconds each, followed by tap waterrinses after each step. Slides were then treated with 5% water inalcohol (2 fresh solutions) then 100% alcohol (3 fresh solutions) thenxylene (3 fresh solutions, 5 minutes each). Finally, a coverslip wasapplied to protect the section. Tumor areas were outlined on theH&E-stained sections by a board-certified pathologist using afine-tipped permanent pen. Section areas for cell line slides weresimilarly outlined. Tumor areas and section areas for cell lines werequantified by image capture on a flat-bed scanner and analyzed usingImagePro software (Media Cybernetics).

The final quantification terms for the target protein detected by theVeraTag assay can be either RPA for similar samples with identical CBvolume or the RPA*CB vol/tumor area to account for different amounts oftumor on the slides. This is generally expressed as RPA*μL/cm². Thereproducibility between samples tested on different days and differentdays compared to the mean is shown in FIG. 5 b. The diagonal in eachplot indicates perfect correlation. To adjust for batch to batchvariability, multiple standard cell lines of expected signal levels areincluded in each batch to facilitate normalization. The results for arange of transfected cell lines with varying amounts of p95, pre- andpost-normalization, for 3 different operators, are shown in FIG. 5 c.Batch to batch normalization is limited to a multiplication by a singlefactor for each batch that achieves a least squares fit of the log ofthe measurement on these standards to the log of their expected values.Expected values of each standard are established by measuring allstandards (typically 3) in a single batch with multiple replicates, withthe replicates running over multiple batches. As new production lots ofeach standard are introduced, the new lot is measured with multiplereplicates against the current set of standards to establish an expectedvalue to be associated with that particular lot.

Results from a series of antibodies from the Series D immunization isshown in FIG. 6, compared against anti-HER2 Ab8. D4.1 and D12.1 behavedmuch like Ab8, indicating that the epitope recognized by theseantibodies is detected equally well in p95 as full-length HER2. D8.1 andD9.1 however show about 10-fold stronger signal from the p95-expressingcell lines (MCF7-p95, MCF7-p95c and SKBR3-p95c) than their parentallines (MCF7 and SKBR3), demonstrating specificity for p95. Even thoughfull-length HER2 contains the same peptide sequence as p95, the epitopesof at least D8.1 and D9.1 are likely hidden in full-length HER2 in theFFPE format. MCF7-HER2c shows a modestly increased signal above MCF7,consistent with the small amount of p95 found in FIG. 4, lane 1.

Example 5 Quantification of p95 in FFPE Slides of Primary Breast CancerTumors

A set of 12 breast cancer tumors were obtained from Asterand (Cohort A).These tumors were chosen to be highly HER2-positive (Allred score of 8),and came from patients with node-positive status. Both of these factorsare known to correlate with p95-positivity. The tumors were prepared byAsterand such that approximately half of the tumor was fresh frozen andhalf was formalin-fixed and paraffin-embedded. This enabled detection ofp95 by Western blot, which should imply p95 expression in FFPE slidescut from the matching portion of tumor.

The frozen portion of the tumors was lysed by grinding under liquidnitrogen with a mortar and pestle until a power was obtained. A minimumamount of cold lysis buffer (about 600 μL) was added and the mortar wasleft on ice for 30 minutes. Lysis Buffer contained 1% Triton X-100, 50mM Tris-HCl (pH7.5), 150 mM NaCl, 50 mM NaF, 50 mM sodiumbeta-glycerophosphate, 1 mM Na₃VO₄, 5 mM EDTA, 10 μg/mL pepstatin and 1tablet Complete Protease Inhibitor (Roche #1836170) in 10 mL water. Thesolution was collected into pre-chilled microfuge tubes and centrifugedat 4° C. for 20 minutes. Aliquots were stored at −80° C. until use.

A Western blot (FIG. 7 a) stained with an antibody raised against theintracellular domain of HER2, CB11 (Ventana), showed that several tumorsexpressed p95 with various levels of expression. Tumors 1-3, 5, 7, 8 and10 were designated as p95 positive while all others were designatedp95-negative. FFPE slides from all 12 tumors along with 7 cell linestandards were tested in the assay described in Example 4, using theclone D9.1 (FIG. 7 b). The distinction between p95-positive andp95-negative FFPE tumors was nearly perfect (replotted in FIG. 7 c),especially considering the possibility of heterogeneity between theportion that was lysed for the Western and that which was fixed for theFFPE p95 assay.

Example 6 Demonstration of Sensitivity by an Isotype Control Experiment

The sensitivity of the FFPE p95 assay was further demonstrated bycomparison with control measurements where the D9.1 antibody was swappedwith an isotype control (FIG. 8 a). For both MCF-7 and the highHER2-expressor SKBR3, the signal generated with D9.1 was not muchdifferent from the signal generated when D9.1 was replaced with itsisotype control. Conversely, when p95 is present (MCF7-p95 transient,MCF7-p95 clone and SKBR3-p95) signals generated in the presence of D9.1far exceeds the isotype control. For the tumors, p95-positive tumorsshowed signals using D9.1 well above those where the isotype control isused whereas most of the p95-negatives showed signals similar to thosewhere D9.1 was absent (FIG. 8 b). FIG. 8 c shows the difference betweenthe signals generated using D9.1 and the isotype control. The differencein the means of p95-positive and p95-negatives is approximately 4-foldwith a dynamic range of approximately 10-fold.

Example 7 Increased Likelihood of p95-Positivity in Samples Measured tobe Highly HER2-Positive by the HERmark® HER2-Total Assay

The VeraTag HER2 total (H2T) assay quantifies the amount of HER2 proteinper unit area of invasive tumor as described in U.S. patent applicationNo. 61/015,608, which is incorporated by reference herein, including anydrawings. As p95 is expected to be more prevalent in tumors that scoreat the high end of HER2-positive by IHC or H2T, the tumors used inExample 5 were tested by the H2T assay to search for a possible cutoff.The tumors were found to span the range of HER2-positivity as assessedby the H2T assay (FIG. 9 a), however the p95-positives weresignificantly higher by H2T than the p95-negatives.

A second set of 18 FFPE tumors (Cohort B) was obtained from Asterand tofurther explore the distribution of p95 within the H2T landscape. Slidesfrom these tumors were assessed by both the p95 assay as described inExample 4 and the H2T assay. Correlative results for both Cohort A and Bare presented in FIG. 9 b. A higher p95 signal is more likely to befound with high H2T for both cohorts.

It was therefore hypothesized that trastuzumab-treated patients with H2Tscores more consistent with the p95-positive tumors measured in Cohort Ashould have worse outcomes than those with lower H2T scores since p95 islacking the trastuzumab epitope and is therefore a likely means ofescape. To test this hypothesis, a cohort of trastuzumab-treatedpatients whose tumors had previously been assessed for H2T wereinvestigated for evidence of poor outcomes in the range of H2T expectedto be enriched in p95. This cohort is further described in U.S. patentapplication No. 61/015,608, which is incorporated by reference hereinincluding any drawings. This cohort (N=92) was derived from theInternational Serum Her2/neu Study Group (ISHSG) and is called theLipton cohort. These patients were selected primarily by IHC performedat a central location—the University of Vienna in Austria—by a singlepathologist. 90% of patients were IHC 3+, and 80/92 received trastuzumabin combination with chemotherapy, while 12 received trastuzumab as asingle drug. 88/92 patients had metastatic breast cancer, and they couldhave received trastuzumab either as first, second or third line therapy.

For these analyses, a cutoff for H2T was chosen just above the highestp95-negative shown in FIG. 9 a, at a value of log₁₀(H2T) 1.95. Abovethis H2T cutoff, tumors could be described as p95-enriched while thosebelow the cutoff would be p95-equivocal. Among the patients confirmed tobe HER2-FISH-positive by central lab analysis, those in the p95-enrichedgroup had significantly shorter time-to-progression (HR=2.0; p=0.017)than those that were in the p95-equivocal group (FIG. 9 c). The overallsurvival results (FIG. 9 d) were similar (HR=1.8; p=0.056). Theseresults suggest that a highly quantitative measure of HER2 expression,such as the VeraTag H2T assay, can identify subpopulations expected tobe enriched in patients with tumors that contain p95 to the degree thattrastuzumab is significantly less effective. This subpopulation wouldthen be candidates for alternative treatments such as HER2 tyrosinekinase inhibitors.

Example 8 Colorimetric Immunohistochemistry of FFPE Cell Lines andTumours with an Anti-p95 Antibody

Parental or p95-CTF-transfected breast tumor cell lines SKBR3 orSKBR3-p95c (˜1×10⁶p185-HER2/cell) were grown, formaldehyde fixed, andprepared for FFPE sections as described in Example 3. Staining for p95or full-length HER2 was performed using the Vectastain ELITE ABCPeroxidase kit (PK 6102), the ImmPact DAB peroxidase substrate (Vector#SK4105), and horse serum from the Vectastain kit. The cells werecounterstained with hematoxylin (Vector #H3401). FFPE cells in paraffinblocks were microtome cut to 5 μm thickness and placed on glass slides.The epitope retrieval process was similar to Example 4. Briefly,sections were deparaffinized by standard sequential xylene, 100% ethanoland 70% ethanol extractions, and epitope retrieval was performed in DIVAbuffer and raised to a boil in a microwave oven (3.5 min at power=10,followed by 10 min at power=3). Following cooling for 1 hour, sectionsare washed 6× with water, blocked with horse serum containing blockingbuffer for 1 hr at room temperature (RT), then incubated with 4 μg/mL ofD9.1 anti-p95 monoclonal antibody in blocking buffer for 1 hour at RT.The sections are washed with PBS and the secondary antibody addition andsubsequent color development and hematoxylin staining steps wereperformed as described in the manufacturer's kit protocol. Cellmicrographs were taken with a digital camera image capture systemmounted on a Leica microscope. The results are shown in FIG. 10.

FIG. 10 a shows staining by the p95-recognizing D9.1 antibody of severalcell lines. In the parental MCF7 cells, which express low levels ofHer2, little staining is observed. In MCF7 cells transfected with a p95expression vector, high levels of staining are seen; however, in cellstransfected with full length Her2 expression vectors, staining is notseen, verifying the specificity of the D9.1 antibody. In the parentalSKBR-3 cells, which express high amounts of full-length HER2 and lowlevels of p95, little cell membranous staining is observed, consistentwith the low level of p95; in contrast, in cells transfected with CTF(C-terminal fragment), which is an intracellular domain of Her2, strongstaining is observed. Control experiments using a human IgG2a isotypecontrol antibody replacing D9.1 (IgG2a isotype) lacked the cellmembranous staining observed for D9.1, indicating specificity of the D9staining (data not shown).

Slides from Cohort A described in Example 5 were used to test theability of D9.1 to detect p95 in FFPE tumors. As described in Example 5,#5 was identified as positive for p95 by Western blot analysis whereas#6 and #12 was negative. FFPE sections were examined for IHC staining byD9.1 or CB-11 antibodies as described above in Example 8, using theVectastain ELITE ABC Peroxidase kit (PK 6102). The cells werecounterstained with hematoxylin (Vector #H3401).

FIG. 10 b represents staining by the p95-recognizing D9.1 antibody ofthe p95-positive #5. Strong cell membranous staining is observed,consistent with p95 expression observed in the Western blot and VeraTagassay. Similar strong membranous staining is observed with CB11,consistent with high expression of p95- and p185-HER2 observed byWestern blotting and HER2 VeraTag assay. FIG. 10 b also shows theabsence of cell membranous staining by D9.1 antibody applied to tumor#12 and #6 and is consistent with the absence of p95 by Western blot andVeraTag assay. However, the significant full-length HER2 levels found byWestern blot and VeraTag assay of #5 is consistent with the strongmembranous staining observed with CB-11, an antibody targeted to theintracellular domain of Her2. Taken together with the cell line controlstaining, the data supports results from the p95 VeraTag assayindicating preferential binding of the D9.1 antibody to p95 compared tofull-length HER2. Furthermore, the data suggest that the amino acidsequence recognized by D9.1 is contained in naturally occurring forms ofp95 expressed in breast tumors. Control experiments using a human IgG2aisotype control antibody replacing D9.1 (IgG2a isotype) lacked the cellmembranous staining observed for D9.1, indicating specificity of theD9.1 staining on tumor tissues (data not shown).

Example 9 Quantitation of p95 Using Directly Labeled Anti-p95 Antibodiesin a VeraTag Assay

In Example 4, p95 was measured in FFPE slides using specific anti-p95antibodies in conjunction with an anti-mouse secondary antibody labeledwith a cleavable vTag. In this example, p95 was measured with directlylabeled p95 antibodies. The assay method is identical to that describedin Example 4 except that the addition of labeled secondary andsubsequent wash was omitted and D9-Pro125 was used at 1.0 μg/mL. Theresults presented in FIG. 11 are similar to those shown in FIG. 6 withpossible reduction in the dynamic range between p95-low/negative celllines (MCF7, MCF7-HER2, SKBR3) and the p95-high cell lines (MCF7-p95c,MCF7-p95 and SKBR3-p95c). The D9.1 antibody was also used in this formatin a batch of cell lines (FIG. 12 a) and tumors from Cohort A (FIG. 12b).

Example 10 Quantitation of p95 Using an Anti-p95 Antibody and anAntibody Directed Against the Intracellular Domain of HER2 in a VeraTagAssay

In this example, the specificity of D9.1 was paired with anti-HER2 Ab8(clone e2-4001) from Labvision in the form of a cleavable VeraTag assayas described in U.S. patent application No. 61/015,608, which isincorporated by reference herein, including any drawings. In thisexample anti-HER2 Ab8 labeled with biotin at 2 μg/mL was used withanti-p95 D9.1 at 2 μg/mL. Results from this form of the assay arepresented in FIGS. 12 c and 12 d. Separation of p95-positive andp95-negatives is retained with this form of the assay.

Example 11 Growth Inhibition of a Breast Cancer Cell Line Using Anti-p95Antibodies

The antibodies from the D-series of immunizations were tested for theirability to inhibit growth of the high-HER2 expressing cell lines SKBR3and BT474. SKBR3 and BT474 cells were plated in ½-area 96-well plates ata density of 120 k/well and 240 k/well, respectively. SKBR3 cells werecultured in 100 μL McCoy's 5A (ATCC) 10% FBS, 1% penicillin-streptomycinand 2 mM L-glutamine. BT474 cells were maintained in 100 μL at 37° C. in5% CO₂ in 50:50 DMEM: F12, 10% FBS, 1% penicillin-streptomycin and 2 mML-glutamine. After cells were allowed to attach for 4 hours, purifiedantibodies listed in FIG. 13 were added to final concentrations of 1.0,3 and 10 μg/mL. 4D5 included as a positive control. The cells wereallowed to grow for 3 days before cell growth was assessed by the XTTassay (Sigma) per the manufacturer's instructions. The difference inabsorbance at 492 nm and 690 nm was taken as proportional to the numberof cells in each well. These results (FIG. 13) suggest that D3.4 and tosome degree D4.1 inhibit the growth of SKBR3 cells but not BT474. Thisdifference in reactivity towards two cells lines with near equally highlevels of HER2 expression may be explained by the fact that SKBR3 isknow to shed greater levels of HER2 extracellular domain into the mediaand therefore may be more dependent on p95 retained in the cell.

Example 12 Very High Levels of her2 Correlates with Poor Response toTrastuzumab

The HERmark® assay was used to measure the total Her2 protein (H2T) perunit area of invasive tumor tissue (as described in U.S. PatentApplication No. 61/015,608, which is incorporated by reference herein,including any drawings) in formalin-fixed, paraffin-embedded (FFPE)primary breast tumor specimens from 99 women treated with trastuzumabfor metastatic breast cancer (MBC). Table 4 shows the characteristics ofthe patient population from which the tumors were derived. Specimenswere also tested by central FISH.

TABLE 4 Patient Characteristics Value Value Characteristic (range, %)Characteristic (range, %) Total Patients 99 Treatment Mean Follow Up32.0 (11.8-72.3) Trastuzumab + 87 (87.9%) (months) chemotherapy Mean Age55.2 (27.6-85.4) Trastuzumab only 12 (12.1%) Line of chemotherapyHormonal Status First line 72 (72.7%) ER+PR+ 15 (15.2%) Second line 17(17.2%) ER+PR− 19 (19.2%) Third line 8 (8.1%) ER−PR+ 3 (3%) Unknown 2(2.0%) ER−PR− 60 (60.06%) Number of metastatic sites Unknown 2 (2.0%) <357 (57.6%) ≧3 42 (42.4%

A sub-population treatment effect pattern plot (STEPP) was generated toexamine the progression-free survival (PFS) rate at 12 months aftertreatment with trastuzumab across the distribution of H2T. Bins of 30patients were ordered smallest to largest H2T. The results are shown inFIG. 14. A trend of increasing probability of remaining progression-freepast 12 months was observed for increasing H2T. However, at the highestlevels of H2T, an abrupt decrease in the PFS rate was observed,consistent with a reduction in susceptibility to trastuzumab.

Kaplan-Meier (KM) analyses were performed comparing the PFS of FISH(−),H2T low (log₁₀H2T<1.25) patients with those of FISH(+), H2T high(Log₁₀H2T≧1.95 and FISH(+), H2T intermediate (1.25<log₁₀H2T<1.95).Cut-offs were identified by lowest p-value in a positional scanninganalysis. KM analyses demonstrated that patients who were FISH(+), H2Tintermediate had a significantly longer PFS than patients who wereFISH(−), H2T low (median PFS 12.6 vs. 4.5 months; hazard ratio(HR)=0.34; p<0.0001). Patients that were FISH(+), H2T high experienced aPFS that was no better than patients that were FISH(−), H2T low (medianPFS 4.6 vs. 4.5 months; HR=0.87; p=0.68). The results of the KM analysesare shown in FIG. 15. The HERmark® assay identified patients with tumorshaving highly over-expressed HER2 and poor performance on trastuzumab.Neither the magnitude of HER2 over-expression nor the outcome for thissubgroup was predictable by FISH/CEP17 copy number. MBC patients withvery high levels of H2T may represent a subset of patients with de novoresistance to trastuzumab.

While the applicants do not wish to be confined to any particularmechanistic theory, possible mechanisms that may account for the poorresponse to trastuzumab observed in this subgroup may include:

-   -   insufficient trastuzumab    -   increased signaling via formation of heterodimers that are not        completely suppressed by trastuzumab    -   generation of C-terminal fragments of Her2 such as HER2p95. Six        of the 15 patients in the very high H2T subgroup were        HER2p95-positive by the p95 VeraTag assay.

BIOLOGICAL DEPOSITS

A deposit of three hybridoma cell lines that produce the monoclonalantibodies referred to herein as p95.D3.4, p95.D8.2 and p95.D9.1 wasmade on Jan. 28, 2009, to the American Type Culture Collection (ATCC,10801 University Blvd., Manassas, Va.) under conditions prescribed bythe Budapest Treaty. The ATCC accession numbers for the depositedhybridoma cell lines are as follows: PTA-9738 (p95.D3.4), PTA-9739(p95.D8.2) and PTA-9740 (p95.D9.1). As required under the BudapestTreaty, the cell lines will be irrevocably and without restriction orcondition released to the public upon the issuance of a patent.

All publications and other materials described herein are used toilluminate the invention or provide additional details respecting thepractice and are incorporated by reference in their entirety.

1-30. (canceled)
 31. A method of producing antibodies specific ap95Her-2 protein fragment having a first amino acid corresponding tomethionine 611 of Her-2 protein (p95Her-2), comprising: a) immunizing amammal with an immunogen comprising a peptide comprising an amino acidsequence set forth in one of SEQ ID NOs 1-7; b) generating one or morehybridoma cell lines expressing monoclonal antibodies from the immunizedmammal; c) screening the one or more hybridoma cell lines to identify ahybridoma cell line that express a monoclonal antibody that bindsspecifically to p95Her-2 but does not bind specifically to full lengthnative Her-2 protein; d) establishing monoclonal cultures for one ormore specific hybridoma cell lines that express an antibody that bindsspecifically to p95Her-2; and e) isolating one or more monoclonalantibodies that bind specifically to p95Her-2 but do not bindspecifically to full length native Her-2 protein from the monoclonalcultures.
 32. The method of claim 31, wherein the peptide of theimmunogen is conjugated to keyhole limpet hemocyanin (KLH).
 33. Themethod of claim 31, wherein the immunizing is performed over a period ofabout 5 weeks.
 34. The method of claim 31, wherein the immunizingcomprises twice-weekly administration of the immunogen to the mammal.35. The method of claim 31, wherein the screening comprises analyzingculture supernatant from the one or more hybridoma cell lines using atleast one of an enzyme-linked immunosorbent assay, Western blotting, orflow cytometry.
 36. The method of claim 35, wherein the screeningcomprises using a peptide having an amino acid sequence as set forth inat least one of SEQ ID Nos:1-7 to screen culture supernatant from theone or more hybridoma cell lines.
 37. The method of claim 36, whereinthe peptide is conjugated to bovine serum albumin (BSA).
 38. The methodof claim 31, wherein the screening comprises analyzing culturesupernatant from the one or more hybridoma cell lines using anenzyme-linked immunosorbent assay for the ability to bind to at leastone of a peptide having an amino acid sequence as set forth in at leastone of SEQ ID Nos:1-7 or full length native Her-2 protein.
 39. Themethod of claim 35, wherein the screening comprises Western blotstaining of cell lysates containing p95Her-2 or Her-2 using culturesupernatant from the one or more hybridoma cell lines of step c). 40.The method of claim 35, wherein the screening comprises contacting cellsexpressing p95Her-2 or Her-2 with culture supernatant from the one ormore hybridoma cell lines of step c) and analyzing the contacted cellsby flow cytometry using a secondary antibody.
 41. The method of claim40, wherein the cells expressing p95Her-2 or Her-2 are cells that havebeen transiently transfected to express p95Her-2 or Her-2.
 42. Themethod of claim 40, wherein the cells expressing p95Her-2 or Her-2 arefixed prior to analyzing by flow cytometry.
 43. The method of claim 31,wherein the amino acid sequence of the peptide comprises SEQ ID NO:5.44. The method of claim 36, wherein the amino acid sequence of thepeptide comprises SEQ ID NO:5.
 45. The method of claim 31, wherein themammal is a mouse or a rabbit.